Chemical modifications of monomers and oligonucleotides with cycloaddition

ABSTRACT

The invention features compounds of formula I or II: 
     
       
         
         
             
             
         
       
     
     In one embodiment, the invention relates compounds and processes for conjugating ligand to oligonucleotide. The invention further relates to methods for treating various disorders and diseases such as viral infections, bacterial infections, parasitic infections, cancers, allergies, autoimmune diseases, immunodeficiencies and immunosuppression.

PRIORITY

This application is a continuation application of U.S. application Ser.No. 13/120,389, filed Oct. 17, 2011, which claims benefit of priority toPCT Application No. PCT/US2009/058084, filed Sep. 23, 2009, which claimspriority to U.S. Application No. 61/099,497, filed on Sep. 23, 2008, allof which are herein incorporated by reference in their entirety.

FIELD OF INVENTION

The present invention relates to the field of conjugation of ligands tooligonucleotides with “click” chemistry.

BACKGROUND

Oligonucleotide compounds have important therapeutic applications inmedicine. Oligonucleotides can be used to silence genes that areresponsible for a particular disease. Gene-silencing prevents formationof a protein by inhibiting translation Importantly, gene-silencingagents are a promising alternative to traditional small, organiccompounds that inhibit the function of the protein linked to thedisease. siRNA, antisense RNA, and micro-RNA are oligonucleotides thatprevent the formation of proteins by gene-silencing.

RNA interference or “RNAi” is a term initially coined by Fire andco-workers to describe the observation that double-stranded RNA (dsRNA)can block gene expression (Fire et al. (1998) Nature 391, 806-811;Elbashir et al. (2001) Genes Dev. 15, 188-200). Short dsRNA directsgene-specific, post-transcriptional silencing in many organisms,including vertebrates, and has provided a new tool for studying genefunction. RNAi is mediated by RNA-induced silencing complex (RISC), asequence-specific, multi-component nuclease that destroys messenger RNAshomologous to the silencing trigger. RISC is known to contain short RNAs(approximately 22 nucleotides) derived from the double-stranded RNAtrigger, but the protein components of this activity remained unknown.

siRNA compounds are promising agents for a variety of diagnostic andtherapeutic purposes. siRNA compounds can be used to identify thefunction of a gene. In addition, siRNA compounds offer enormouspotential as a new type of pharmaceutical agent which acts by silencingdisease-causing genes. Research is currently underway to developinterference RNA therapeutic agents for the treatment of many diseasesincluding central-nervous-system diseases, inflammatory diseases,metabolic disorders, oncology, infectious diseases, and ocular disease.

Many diseases (e.g., cancers, hematopoietic disorders, endocrinedisorders, and immune disorders) arise from the abnormal or otherwiseunwanted expression or activity of a particular gene or group of genes.For example, disease can result through misregulated gene expression,expression of a mutant form of a protein, or expression of viral,bacterial or other pathogen-derived genes. The RNAi pathway can be usedto inhibit or decrease the unwanted expression of such genes (Agrawal etal., Microbiol Mol Biol Rev., 2003, 67, 657-685; Alisky & Davidson, Am.J. Pharmacogenomics, 2004, 4, 45-51).

In yet a further aspect, the invention relates to a method for treatinga disease or disorder in a subject. The method includes identifying asubject having or at risk for developing the disease, administering apharmaceutical composition containing an immunoselective iRNA agenthaving one or more of the modified nucleotides or linkages describedabove, and a pharmaceutically acceptable carrier. The subject may bemonitored for an effect on the immune system, e.g., an immunostimulatoryor immunoinhibitory response, such as by monitoring for increasedexpression of a growth factor, such as a cytokine or a cell-surfacereceptor (e.g., a Toll-like receptor) as described above. Cytokines ofinterest can be those expressed from T cells, B cells, monocytes,macrophages, dendritic cells, or natural killer cells of the subject.The assays can be performed using blood or serum samples from thesubject. The disease or disorder can be one where it is particularlyundesirable to stimulate the immune system, e.g., in a patient that hasreceived organ, tissue or bone marrow transplants. In anotheralternative, the disease or disorder can be one where it is particularlydesirable to stimulate the immune system, e.g., in patients with canceror viral diseases. In one embodiment, the subject is immunocompromised,and an iRNA agent that includes nucleotide modifications stimulates animmune response in a cell to a greater extent than an iRNA agent thatdoes not include nucleotide modifications. The subject may be a mammal,such as a human.

In a preferred embodiment, administration of an immunoselective iRNAagent is for treatment of a disease or disorder present in the subject.In another preferred embodiment, administration of the iRNA agent is forprophylactic treatment.

It is therefore an object of the present invention to providepolynucleotides/oligonucleotides which are capable of stimulating ananti-viral response, in particular, a type I IFN response. It is anotherobject of the present invention to provide a pharmaceutical compositioncapable of inducing an anti-viral response, in particular, type I IFNproduction, in a patient for the prevention and treatment of diseasesand disorders such as viral infection. It is also an object of thepresent invention to provide a pharmaceutical composition for treatingtumor.

The disease and/or disorder include, but are not limited to infections,tumor, allergy, multiple sclerosis, and immune disorders.

Infections include, but are not limited to, viral infections, bacterialinfections, anthrax, parasitic infections, fungal infections and prioninfection. Viral infections include, but are not limited to, infectionby hepatitis C, hepatitis B, herpes simplex virus (HSV), HIV-AIDS,poliovirus, encephalomyocarditis virus (EMCV) and smallpox virus.Examples of (+) strand RNA viruses which can be targeted for inhibitioninclude, without limitation, picornaviruses, caliciviruses, nodaviruses,coronaviruses, arteriviruses, flaviviruses, and togaviruses. Examples ofpicornaviruses include enterovirus (poliovirus 1), rhinovirus (humanrhinovirus 1A), hepatovirus (hepatitis A virus), cardiovirus(encephalomyocarditis virus), aphthovirus (foot-and-mouth disease virusO), and parechovirus (human echovirus 22). Examples of calicivirusesinclude vesiculovirus (swine vesicular exanthema virus), lagovirus(rabbit hemorrhagic disease virus), “Norwalk-like viruses” (Norwalkvirus), “Sapporo-like viruses” (Sapporo virus), and “hepatitis E-likeviruses” (hepatitis E virus). Betanodavirus (striped jack nervousnecrosis virus) is the representative nodavirus. Coronaviruses includecoronavirus (avian infections bronchitis virus) and torovirus (Bernevirus). Arterivirus (equine arteritis virus) is the representativearteriviridus. Togavirises include alphavirus (Sindbis virus) andrubivirus (Rubella virus). Finally, the flaviviruses include flavivirus(Yellow fever virus), pestivirus (bovine diarrhea virus), andhepacivirus (hepatitis C virus).

In certain embodiments, the viral infections are selected from chronichepatitis B, chronic hepatitis C, HIV infection, RSV infection, HSVinfection, VSV infection, CMV infection, and influenza infection.

In one embodiment, the infection to be prevented and/or treated is upperrespiratory tract infections caused by viruses and/or bacteria. Inanother embodiment, the infection to be prevented and/or treated is birdflu.

Bacterial infections include, but are not limited to, streptococci,staphylococci, E. coli, pseudomonas.

In one embodiment, bacterial infection is intracellular bacterialinfection. Intracellular bacterial infection refers to infection byintracellular bacteria such as mycobacteria (tuberculosis), chlamydia,mycoplasma, listeria, and facultative intracellular bacteria such asstaphylococcus aureus.

Parasitic infections include, but are not limited to, worm infections,in particular, intestinal worm infection.

Tumors include both benign and malignant tumors (i.e., cancer).

Cancers include, but are not limited to biliary tract cancer, braincancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer,endometrial cancer, esophageal cancer, gastric cancer, intraepithelialneoplasm, leukemia, lymphoma, liver cancer, lung cancer, melanoma,myelomas, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer,prostate cancer, rectal cancer, sarcoma, skin cancer, testicular cancer,thyroid cancer and renal cancer.

In certain embodiments, cancers are selected from hairy cell leukemia,chronic myelogenous leukemia, cutaneous T-cell leukemia, chronic myeloidleukemia, non-Hodgkin's lymphoma, multiple myeloma, follicular lymphoma,malignant melanoma, squamous cell carcinoma, renal cell carcinoma,prostate carcinoma, bladder cell carcinoma, breast carcinoma, ovariancarcinoma, non-small cell lung cancer, small cell lung cancer,hepatocellular carcinoma, basaliom, colon carcinoma, cervical dysplasia,and Kaposi's sarcoma (AIDS-related and non-AIDS related).

Allergies include, but are not limited to, respiratory allergies,contact allergies and food allergies.

Immune disorders include, but are not limited to, autoimmune diseases,immunodeficiency, and immunosuppression.

Autoimmune diseases include, but are not limited to, diabetes mellitus,arthritis (including rheumatoid arthritis, juvenile rheumatoidarthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis,encephalomyelitis, myasthenia gravis, systemic lupus erythematosis,automimmune thyroiditis, dermatitis (including atopic dermatitis andeczematous dermatitis), psoriasis, Sjogren's Syndrome, Crohn's disease,aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerativecolitis, asthma, allergic asthma, cutaneous lupus erythematosus,scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversalreactions, erythema nodosum leprosum, autoimmune uveitis, allergicencephalomyelitis, acute necrotizing hemorrhagic encephalopathy,idiopathic bilateral progressive sensorineural hearing, loss, aplasticanemia, pure red cell anemia, idiopathic thrombocytopenia,polychondritis, Wegener's granulomatosis, chronic active hepatitis,Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves'disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior, andinterstitial lung fibrosis. Immunodeficiencies include, but are notlimited to, spontaneous immunodeficiency, acquired immunodeficiency(including AIDS), drug-induced immunodeficiency (such as that induced byimmunosuppressants used in transplantation and chemotherapeutic agentsused for treating cancer), immunosuppression caused by chronichemodialysis, trauma or surgical procedures.

Immunosuppression includes, but is not limited to, bone marrowsuppression by cytotoxic chemotherapy.

siRNA has been shown to be extremely effective as a potential anti-viraltherapeutic with numerous published examples appearing recently. siRNAmolecules directed against targets in the viral genome dramaticallyreduce viral titers by orders of magnitude in animal models of influenza(Ge et al., (2004) Proc. Natl. Acd. Sci. USA, 101, 8676-8681; Tompkinset al. (2004) Proc. Natl. Acd. Sci. USA, 101, 8682-8686; Thomas et al.(2005) Expert Opin. Biol. Ther. 5, 495-505), respiratory synctial virus(RSV) (Bitko et al. (2005) Nat. Med. 11, 50-55), hepatitis B virus (HBV)(Morrissey et al. (2005) Nat. Biotechnol. 23, 1002-1007), hepatitis Cvirus (Kapadia et al. (2003) Proc. Natl. Acad. Sci. USA, 100, 2014-2018;Wilson et al. (2003) Proc. Natl. Acad. Sci. USA, 100, 2783-2788) andSARS coronavirus (Li et al. (2005) Nat. Med. 11, 944-951).

The opportunity to use these and other nucleic acid based therapiesholds significant promise, providing solutions to medical problems thatcould not be addressed with current, traditional medicines. The locationand sequences of an increasing number of disease-related genes are beingidentified, and clinical testing of nucleic acid-based therapeutics fora variety of diseases is now underway.

Despite the different synthetic strategies developed for conjugation ofvarious ligands to the oligonucleotides, the synthesis ofligand-oligonucleotide conjugates is anything but trivial and requiresextensive expertise in organic chemistry and solid-phase synthesis. Areal advance would be to use a coupling reaction that can be utilizedfor a large variety of ligands and oligonucleotides. The Huisgen1,3-dipolar cycloaddition of alkynes and azides, the “click” reaction,is especially attractive for irreversible coupling of two moleculesunder mild conditions. The “click” chemistry has recently emerged as anefficient strategy to conjugate carbohydrates, peptides and proteins,fluorescent labels and lipids to oligonucleotides. Therefore, there is aclear need for new reagents that can be utilize for “click” chemistryfor conjugation of ligands to oligonucleotides. The present invention isdirected to this very important end.

SUMMARY

The invention relates to compounds that can be used as a ribosereplacement or can be used as universal base to conjugate variousligands to oligonucleotides, e.g. iRNA agents, through “click”chemistry. These compounds are also referred to as the “click-carrier”herein.

For instance, the invention features a compound having the structureshown in formula (I)

wherein:

X is O, S, NR^(N) or CR^(P) ₂;

B is independently for each occurrence hydrogen, optionally substitutednatural or non-natural nucleobase, optionally substituted triazole oroptionally substituted tetrazole; NH—C(O)—O—C(CH₂B₁)₃,NH—C(O)—NH—C(CH₂B₁)₃; where B₁ is halogen, mesylate, N₃, CN, optionallysubstituted triazole or optionally substituted tetrazole;

R¹, R², R³, R⁴ and R⁵ are each independently for each occurrence H, OR⁶,F, N(R^(N))₂, N₃, CN, -J-linker-N₃, -J-linker-CN, -J-linker-C≡R⁸,-J-linker-cycloalkyne, -J-linker-R_(L), or -J-linker-Q-linker-R^(L);

J is absent, O, S, NR^(N), OC(O)NH, NHC(O)O, C(O)NH, NHC(O), NHSO,NHSO₂, NHSO₂NH, OC(O), C(O)O, OC(O)O, NHC(O)NH, NHC(S)NH, OC(S)NH,OP(N(R^(P))₂)O, or OP(N(R^(P))₂);

R⁶ is independently for each occurrence hydrogen, hydroxyl protectinggroup, optionally substituted alkyl, optionally substituted aryl,optionally substituted cycloalkyl, optionally substituted aralkyl,optionally substituted alkenyl, optionally substituted heteroaryl,polyethyleneglycol (PEG), a phosphate, a diphosphate, a triphosphate, aphosphonate, a phosphonothioate, a phosphonodithioate, aphosphorothioate, a phosphorothiolate, a phosphorodithioate, aphosphorothiolothionate, a phosphodiester, a phosphotriester, anactivated phosphate group, an activated phosphite group, aphosphoramidite, a solid support, —P(Z¹)(Z²)—O-nucleoside,—P(Z¹)(Z²)—O-oligonucleotide, —P(Z¹)(Z²)-formula (I),—P(Z¹)(O-linker-Q-linker-R^(L))—O-nucleoside,P(Z¹)(O-linker-R^(L))—O-nucleoside, —P(Z¹)(O-linker-N₃)—O-nucleoside,P(Z¹)(O-linker-CN)—O-nucleoside, P(Z¹)(O-linker-C≡R⁸)—O-nucleoside,P(Z¹)(O-linker-cycloalkyne)-O-nucleoside,—P(Z¹)(O-linker-Q-linker-R^(L))—O-oligonucleotide,P(Z¹)(O-linker-R^(L))—O-oligonucleotide,P(Z¹)(O-linker-N₃)—O-oligonucleotide,—P(Z¹)(O-linker-CN)—O-oligonucleotide,P(Z¹)(O-linker-C≡R⁸)—O-oligonucleotide,P(Z¹)(O-linker-cycloalkyne)-O-oligonucleotide,—P(Z¹)(-linker-Q-linker-R^(L))—O-nucleoside,—P(Z¹)(-linker-Q-R^(L))—O-nucleoside, —P(Z¹)(-linker-N₃)—O-nucleoside,P(Z¹)(-linker-CN)—O-nucleoside, P(Z¹)(-linker-C≡R⁸)—O-nucleoside,P(Z¹)(-linker-cycloalkyne)-O-nucleoside,—P(Z¹)(-linker-Q-linker-R^(L))—O-oligonucleotide,—P(Z¹)(-linker-R^(L))—O-oligonucleotide,P(Z¹)(-linker-N₃)—O-oligonucleotide,—P(Z¹)(-linker-CN)—O-oligonucleotide,P(Z¹)(-linker-C≡R⁸)—O-oligonucleotide orP(Z¹)(-linker-cycloalkyne)-O-oligonucleotide;

R^(N) is independently for each occurrence H, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted cycloalkyl,optionally substituted aralkyl, optionally substituted heteroaryl or anamino protecting group;

R^(P) is independently for each occurrence H, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted cycloalkyl oroptionally substituted heteroaryl;

Q is absent or independently for each occurrence

R^(L) is hydrogen or a ligand;

R⁸ is N or CR⁹;

R⁹ is H, optionally substituted alkyl or silyl;

Z¹ and Z² are each independently for each occurrence O, S or optionallysubstituted alkyl; a

provided that at least one of R¹, R², R³, R⁴ and R⁵ is-J-Linker-Q-Linker-R^(L) or -Linker-Q-R^(L) when B is an unsubstitutednatural base.

In one aspect, the invention features, a compound having the structureshown in formula (Ia)

wherein T₁ and T₂ are each independently H, C₁-C₉ alkyl, C₂-C₉ alkenyl,C₂-C₉ alkynyl, substituted C₁-C₉ alkyl, substituted C₁-C₉ alkenyl andsubstituted C₂-C₉ alkynyl, and R1, R2, R3, R4 R5, X and B are aspreviously defined.

In one embodiment, the invention features, a compound having thestructure shown in formula (II)

A and B are independently for each occurrence hydrogen, protectinggroup, optionally substituted aliphatic, optionally substituted aryl,optionally substituted heteroaryl, polyethyleneglycol (PEG), aphosphate, a diphosphate, a triphosphate, a phosphonate, aphosphonothioate, a phosphonodithioate, a phosphorothioate, aphosphorothiolate, a phosphorodithioate, a phosphorothiolothionate, aphosphodiester, a phosphotriester, an activated phosphate group, anactivated phosphite group, a phosphoramidite, a solid support,—P(Z¹)(Z²)—O-nucleoside, or —P(Z¹)(Z²)—O-oligonucleotide; wherein Z¹ andZ² are each independently for each occurrence O, S or optionallysubstituted alkyl;

J₁ and J₂ are independently O, S, NR^(N), optionally substituted alkyl,OC(O)NH, NHC(O)O, C(O)NH, NHC(O), OC(O), C(O)O, OC(O)O, NHC(O)NH,NHC(S)NH, OC(S)NH, OP(N(R^(P))₂)O, or OP(N(R^(P))₂);

is cyclic group or acyclic group; preferably, the cyclic group isselected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl,imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl,isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl,quinoxalinyl, pyridazinonyl, tetrahydrofuryl and decalin; preferably,the acyclic group is selected from serinol backbone or diethanolaminebackbone;

Q is independently for each occurrence

and

L₁₀ and L₁₁ are independently absent or a linker.

In another embodiment of the present invention there are disclosedpharmaceutical compositions comprising a therapeutically effectiveamount of an iRNA agent of the invention in combination with apharmaceutically acceptable carrier or excipient. In yet anotherembodiment of the invention describes process for preparing saidcompounds

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematics of representative click-conjugate of single strandedoligonucleotides. Multiple conjugation to the 5′-end and allcombinations of multiples are possible with the linker scaffold designand thus conjugation of ligand. By choosing chemistry and order ofconjugation hetero-ligand (separate ligands or hybrid ligand)conjugation are done.

FIG. 2. Schematics of representative click-conjugate of double strandedoligonucleotides. Multiple conjugation to the 5′-end and allcombinations of multiples are possible with the linker scaffold designand thus conjugation of ligand. By choosing chemistry and order ofconjugation hetero-ligand (separate ligands or hybrid ligand)conjugation are done.

FIG. 3. Ring strained alkynes for copper-free click chemistry.

FIG. 4. 3′-Conjugation of ligand to antagomirs, antisenseoligonucleotides and siRNAs using Click Chemistry. The alkyne used isopen chain or cyclic constrained. Cyclic constrained used for clickreaction in the absence of copper. Path 1. Conjugation of ligand-azideto solid support followed by solid phase synthesis, deprotection andpurification of oligonucleotide. Path 2. Conjugation of ligand-azide tosolid-bound oligonucleotide, followed by deprotection and purification.Path 3. Post-synthetic conjugation of ligand-azide to an alkyne bearingoligonucleotide. Annealing with complementary strand gives the siRNA.Direct use of the conjugated single strand enable antagomir andantisense application of the conjugates. L is linker scaffold X is O orS, R is ligand and p is zero or 3-8 with or without substitution on thering carbon. The ring also can have hetero atoms such as N, S, O, SO,SO₂. Substitution on the ring carbon are: single or geminaldisubstitution with F, OMe, COQ where Q is OH, NH₂, NHMe, NMe₂, COOMe,NH(Alkyl), perfluorosubstitution. The site tethering of the cyclicalkyne could be a nitrogen atom as described by Sletten and Bertoszzi inOrganic Letters, 2008, 10, 3097-99. Reverse conjugation of Alkyne tosolid bound azide is another approach or reverse conjugation can beachieved via post-synthesis. In some instance the other regeo isomer isexpected as the major or sole products depends on the nature of theincoming azide and nature of the alkyne. In another instance the productcould be an equimolar mixture of the two regeoselective products thatcould form from the same reaction. Relevant reference for using ringstrained cyclic alkynes for copper-free click chemistry include: Johnsonet al., Copper-free click chemistry for the in situ cross-linking ofphotodegradable star polymers. Chemical Communications, 2008, (26),3064-3066; Sletten and Bertoszzi in Organic Letters, A hydrophilicazacyclooctyne for Cu-free click chemistry, 2008, 10, 3097-99; Baskin etal., Copper-free chlick chemistry for dynamic in vivo imaging. Proc.Natl. Acad. Sci. USA, 2007, 104(43), 16793; Agard et al., A comparativestudy of bioorthogonal reactions with azides., ACS Chem. Biol., 2006, 1,644 and Chen, Fish 'n clicks, Nature Chemical Biology, 2008, 4(7),391-92.

FIG. 5. 5′-Conjugation of ligand to antagomirs, antisenseoligonucleotides and siRNAs using Click Chemistry. The alkyne used isopen chain of cyclic constrained. Cyclic constrained used for clickreaction in the absence of copper. Path 1. Conjugation of ligand-azideto solid support bound oligonucleotide followed by deprotection andpurification of oligonucleotide. Path 2. Post-synthetic conjugation ofligand-azide to an alkyne bearing oligonucleotide. Annealing withcomplementary strand gives the siRNA. Direct use of the conjugatedsingle strand enable antagomir and antisense application of theconjugates. See FIG. 4 for detailed description.

FIG. 6. Conjugation of ligand to antagomirs, antisense oligonucleotidesand siRNAs using abasic linker and click chemistry. Conjugation ofligand to antagomirs, antisense oligonucleotides and siRNAs using abasiclinker and click chemistry. R is the ligand, the alkyne used is openchain of cyclic constrained. Cyclic constrained used for click reactionin the absence of copper. See legend for FIG. 4 for details.

FIG. 7: Schematic representation of ligand attachment points on siRNAs.

FIG. 8: Schematic representation of ligand attachment points onoligonucleotides.

FIG. 9: Conjugation strategies for conjugation of ligands tooligonucleotides by Click Chemistry.

FIG. 10: An abasic linker for Click chemistry.

FIG. 11: An abasic linker for Click chemistry.

FIG. 12: HPLC analysis of ligand conjugation by Click chemistry.

FIG. 13: HPLC analysis of purified Click chemistry conjugation.

FIG. 14: Schematic representation of an Azido-labeled oligonucleotide.

FIG. 15: Schematic representation 6-Carboxyfluorescein-propargylamideconjugated with an azido-labeled oligonucleotide by Click chemistry.

FIG. 16: LC-MS analysis of crude product of Click chemistry reactionbetween 6-Carboxyfluorescein-propargylamide and azide-labeledoligonucleotide.

FIG. 17: Reverse-phase HPLC analysis of click reaction of free spermineazide with RNA alkyne.

FIG. 18: Reverse-phase HPLC (a) and LC-MS analysis of crude reactionmixture of conjugation of Boc-protected spermine-azide with RNA alkyne.

FIG. 19: Schematic representation of special amidites and CPGs used tomake the modified RNA sequences of Table 6.

FIG. 20: Schematic representation of special amidites and CPGs used tomake the RNA-alkyne scaffold. Sequences incorporating these monomers areshown in Table 7.

FIG. 21: FIG. 21 (a) is a schematic representation of exemplary azidesused for click reaction to make the modified RNA sequences of Tables 8and 9, and (b) is a schematic representation of monomers after clickreaction.

FIG. 22: Schematic representation of modified monomers afterincorporation into oligonucleotides. Sequences incorporating thesemonomers are shown in Table 10.

FIG. 23: (a) Schematic representation of modified RNA. (b) and (c)RP-HPLC analysis of purified click products (refer to Table 11)(analysis condition: ¹ Reaction completion was measured by RP-HPLC(DeltaPak C4 column, 150×3.9 mm I.D., 5 μm, 300 Å; buffer A: 50 mM TEAA,pH 7.0; buffer B: ACN; gradient: 0-70% buffer B in 24 min; 30° C., 1mL/min)

FIG. 24: Dose response curves of selected modified siRNAs.

DETAILED DESCRIPTION

In one embodiment of the compounds of the present invention arecompounds represented by formula I or II as illustrated above, or apharmaceutically acceptable salt, ester or prodrug thereof.

Alternatively, formula I, or pharmaceutically acceptable salts orprodrugs thereof, may be represented as:

In one embodiment, X is O, S, NR^(N) or CR^(P) ₂.

In one embodiment, B is hydrogen, optionally substituted natural ornon-natural nucleobase, optionally substituted triazole, or optionallysubstituted tetrazole, NH—C(O)—O—C(CH₂B₁)₃, NH—C(O)—NH—C(CH₂B₁)₃; whereB₁ is independently halogen, mesylate, N₃, CN, optionally substitutedtriazole or optionally substituted tetrazole, and where the nucleobasemay further be substituted by -J-linker-N₃, -J-linker-CN,-J-linker-C≡R⁸, -J-linker-cycloalkyne, -J-linker-R^(L), -Linker-Q-R^(L),or -J-linker-Q-linker-R^(L). B₁ can also be hydrogen.

In one embodiment, R¹, R², R³, R⁴ and R⁵ are each independently H, OR⁶,F, N(R^(N))₂, N₃, CN, -J-linker-N₃, -J-linker-CN, -J-linker-C≡R⁸,-J-linker-cycloalkyne, -J-linker-R^(L), -Linker-Q-R^(L), or-J-linker-Q-linker-R^(L). R¹, R², R³, R⁴ and R⁵ can each also beindependently R⁶.

In one embodiment, J is absent, O, S, NR^(N), OC(O)NH, NHC(O)O, C(O)NH,NHC(O), NHSO, NHSO₂, NHSO₂NH, OC(O), C(O)O, OC(O)O, NHC(O)NH, NHC(S)NH,OC(S)NH, OP(N(R^(P))₂)O, or OP(N(R^(P))₂).

In one embodiment, R⁶ is hydrogen, hydroxyl protecting group, optionallysubstituted alkyl, optionally substituted aryl, optionally substitutedcycloalkyl, optionally substituted aralkyl, optionally substitutedalkenyl, optionally substituted heteroaryl, polyethyleneglycol (PEG), aphosphate, a diphosphate, a triphosphate, a phosphonate, aphosphonothioate, a phosphonodithioate, a phosphorothioate, aphosphorothiolate, a phosphorodithioate, a phosphorothiolothionate, aphosphodiester, a phosphotriester, an activated phosphate group, anactivated phosphite group, a phosphoramidite, a solid support,—P(Z¹)(Z²)—O-nucleoside, —P(Z¹)(Z²)—O-oligonucleotide,—P(Z¹)(Z²)-formula (I), —P(Z¹)(O-linker-Q-linker-R^(L))—O-nucleoside,P(Z¹)(O-linker-R^(L))—O-nucleoside, —P(Z¹)(O-linker-N₃)—O-nucleoside,P(Z¹)(O-linker-CN)—O-nucleoside, P(Z¹)(O-linker-C≡R⁸)—O-nucleoside,P(Z¹)(O-linker-cycloalkyne)-O-nucleoside,—P(Z¹)(O-linker-Q-linker-R^(L))—O-oligonucleotide,P(Z¹)(O-linker-R^(L))—O-oligonucleotide,P(Z¹)(O-linker-N₃)—O-oligonucleotide,—P(Z¹)(O-linker-CN)—O-oligonucleotide,P(Z¹)(O-linker-C≡R⁸)—O-oligonucleotide,P(Z¹)(O-linker-cycloalkyne)-O-oligonucleotide,—P(Z¹)(-linker-Q-linker-R^(L))—O-nucleoside,—P(Z¹)(-linker-Q-R^(L))—O-nucleoside, —P(Z¹)(-linker-N₃)—O-nucleoside,P(Z¹)(-linker-CN)—O-nucleoside, P(Z¹)(-linker-C≡R⁸)—O-nucleoside,P(Z¹)(-linker-cycloalkyne)-O-nucleoside,—P(Z¹)(-linker-Q-linker-R^(L))—O-oligonucleotide,—P(Z¹)(-linker-R^(L))—O-oligonucleotide,P(Z¹)(-linker-N₃)—O-oligonucleotide,—P(Z¹)(-linker-CN)—O-oligonucleotide,P(Z¹)(-linker-C≡R⁸)—O-oligonucleotide orP(Z¹)(-linker-cycloalkyne)-O-oligonucleotide.

In one embodiment, R^(N) is H, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted aryl, optionally substituted cycloalkyl, optionallysubstituted aralkyl, optionally substituted heteroaryl, or an aminoprotecting group.

In one embodiment, R^(P) is independently H, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted cycloalkyl, oroptionally substituted heteroaryl.

In one embodiment, Q is

In one embodiment, R^(L) is hydrogen or a ligand.

In one embodiment, R⁸ is N or CR⁹.

In one embodiment, R⁹ is H, optionally substituted alkyl, or silyl.

In one embodiment, Z¹ and Z² are each independently O, S, or optionallysubstituted alkyl.

In this embodiment of formula I, Q, —N₃, —CN, —CR⁸, an optionallysubstituted triazole, or an optionally substituted tetrazole must bepresent at least once in the compound. For instance, R¹ is, in at leastone instance, N₃, CN, -J-linker-N₃, -J-linker-CN, -J-linker-C≡R⁸,-Linker-Q-R^(L), or -J-linker-Q-linker-R^(L); R² is, in at least oneinstance, N₃, CN, -J-linker-N₃, -J-linker-CN, -J-linker-C≡R⁸,-Linker-Q-R^(L), or -J-linker-Q-linker-R^(L); R³ is, in at least oneinstance, N₃, CN, -J-linker-N₃, -J-linker-CN, -J-linker-C≡R⁸,-Linker-Q-R^(L), or -J-linker-Q-linker-R^(L); R⁴ is, in at least oneinstance, N₃, CN, -J-linker-N₃, -J-linker-CN, -J-linker-C≡R⁸,-Linker-Q-R^(L), or -J-linker-Q-linker-R^(L); R⁵ is, in at least oneinstance, N₃, CN, -J-linker-N₃, -J-linker-CN, -J-linker-C≡R⁸,-Linker-Q-R^(L), or -J-linker-Q-linker-R^(L); B is, in at least oneinstance, optionally substituted triazole, or optionally substitutedtetrazole, NH—C(O)—O—C(CH₂B₁)₃, NH—C(O)—NH—C(CH₂B₁)₃, where B₁ is N₃,CN, optionally substituted triazole or optionally substituted tetrazole,and where the nucleobase is further substituted by -J-linker-N₃,-J-linker-CN, -J-linker-C≡R⁸, -Linker-Q-R^(L), or-J-linker-Q-linker-R^(L); or a combination of the above. In oneembodiment, R¹ is -J-Linker-Q-Linker-R^(L) or -Linker-Q-R^(L). Thus, inone embodiment, R² is -J-Linker-Q-Linker-R^(L) or -Linker-Q-R^(L); inone embodiment, R³ is -J-Linker-Q-Linker-R^(L) or -Linker-Q-R^(L); inone embodiment, R⁴ is -J-Linker-Q-Linker-R^(L) or -Linker-Q-R^(L); inone embodiment, R⁵ is -J-Linker-Q-Linker-R^(L) or -Linker-Q-R^(L); andin one embodiment, B comprises -J-Linker-Q-Linker-R^(L) or-Linker-Q-R^(L).

In another embodiment, the carrier is a ribose sugar as shown in formula(III). In this embodiment, the compound, e.g., a click-carrier compoundis a nucleoside/nucleotide or a nucleoside/nucleotide analog.

In one embodiment, the R² and R⁴ of the same compound are connectedtogether to form a “locked” compound similar to a locked nucleic acid(LNA).

In one embodiment, R¹ and R⁴ are H.

In one embodiment, R¹ is —O-Linker-Q-Linker-R^(L),—OC(O)N(R^(N))-Linker-Q-Linker-R^(L) or -Linker-Q-Linker-R^(L), B is H.

In one embodiment, the R² and R⁴ of the same compound are connectedtogether to form a “locked” compound similar to a locked nucleic acid(LNA).

In one embodiment, when B is hydrogen, R¹ is —O-Linker-Q-Linker-R^(L),—OC(O)N(R⁷)-Linker-Q-Linker-R^(L) or -Linker-Q-Linker-R^(L).

In one embodiment, B is H.

In one embodiment, B is pyrimidine substituted at C5 position.

In one embodiment, R² is OR⁶ and R³ is —O-Linker-Q-Linker-R^(L),—OC(O)N(R^(N))-Linker-Q-Linker-R^(L) or -Linker-Q-Linker-R^(L) and R^(L)is present.

In one embodiment, R³ is OR⁶ and R² is —O-Linker-Q-Linker-R^(L),—OC(O)N(R^(N))-Linker-Q-Linker-R^(L) or -Linker-Q-Linker-R^(L) and R^(L)is present.

In one embodiment, R² is OH.

In one embodiment, R⁹ is H.

In one embodiment, R⁵ is —O-Linker-Q-Linker-R^(L),—OC(O)N(R^(N))-Linker-Q-Linker-R^(L) or -Linker-Q-Linker-R^(L) and R^(L)is present.

In one embodiment, R⁵ is —OC(O)NH(CH₂)_(f)C≡CR⁹, and f is 1-20.

In one embodiment, R⁴ is —OC(O)NH(CH₂)_(f)C≡CR⁹, and f is 1-20.

In one embodiment, R³ is —OC(O)NH(CH₂)_(f)C≡CR⁹, and f is 1-20.

In one embodiment, R² is —OC(O)NH(CH₂)_(f)C≡CR⁹, and f is 1-20.

In one embodiment, R¹ is —OC(O)NH(CH₂)_(f)C≡CR⁹, and f is 1-20.

In one embodiment, B is a nucleobase substituted with—OC(O)NH(CH₂)_(f)C≡CH, and f is 1-20.

In one embodiment, R⁵ is

and f is 1-20.

In one embodiment, R⁴ is

and f is 1-20.

In one embodiment, R³ is

and f is 1-20.

In one embodiment, R² is

and f is 1-20.

In one embodiment, R¹ is

and f is 1-20.

In one embodiment, B is a nucleobase substituted with

and f is 1-20.

In one embodiment, B is a nucleobase substituted with

In one embodiment, B is a nucleobase substituted with

In one embodiment, R⁵ is —O—(CH₂)_(f)C≡CR⁹, and f is 1-20.

In one embodiment, R⁴ is —O—(CH₂)_(f)C≡CR⁹, and f is 1-20.

In one embodiment, R³ is —O—(CH₂)_(f)C≡CR⁹, and f is 1-20.

In one embodiment, R² is —O—(CH₂)_(f)C≡CR⁹, and f is 1-20.

In one embodiment, R¹ is —O—(CH₂)_(f)C≡CR⁹, and f is 1-20.

In one embodiment, B is a nucleobase substituted with —O—(CH₂)_(f)C≡CH,and f is 1-20

In one embodiment, R⁵ is

and f is 1-20.

In one embodiment, R⁴ is

and f is 1-20.

In one embodiment, R³ is

and f is 1-20.

In one embodiment, R² is

and f is 1-20.

In one embodiment, R¹ is

and f is 1-20.

In one embodiment, B is nucleobase substituted with

and f is 1-20.

In one embodiment, f is 1, 2, 3, 4 or 5. In a preferred embodiment, f is1.

In one embodiment Q is

In one embodiment, Q is

In one embodiment, Q is

In one embodiment, Q is

In one embodiment, the ribose sugar of formula (I) has the structureshown in formula (I′).

wherein variable are as defined above for formula (I).In one embodiment, the ribose sugar of formula (I) has the structureshown in formula (I′a).

wherein T₁ and T₂ are each independently H, C₁-C₉ alkyl, C₂-C₉ alkenyl,C₂-C₉ alkynyl, substituted C₁-C₉ alkyl, substituted C₁-C₉ alkenyl andsubstituted C₂-C₉ alkynyl; and R1, R2, R3, R4 R5, X and B are aspreviously defined.

In one embodiment, the ribose sugar of formula (I) has the structureshown in formula (I″).

wherein variable are as defined above for formula (I).

In one embodiment, the ribose sugar of formula (I) has the structureshown in formula (II′a).

wherein T₁ and T₂ are each independently H, C₁-C₉ alkyl, alkenyl, C₂-C₉alkynyl, substituted C₁-C₉ alkyl., substituted C₁-C₉ alkenyl andsubstituted C₂-C₉ alkynyl: and R1, R2, R3, R4 R5, X and B are aspreviously defined.

In one embodiment, when click-carrier compound of formula (I) is at the5′-terminal end of an oligonucleotide, the oligonucleotides is linked atthe R⁵ position of the click-carrier compound.

In one embodiment, when click-carrier compound of formula (I) is at the3′-terminal end of an oligonucleotide, the oligonucleotides is linked atthe R³ or R² position of the click-carrier compound.

In one embodiment, when the click-carrier compound of formula (I) is notat a terminal position in an oligonucleotide, the R⁵ position of thecompound is linked to the 3′- or 2′-position of an oligonucleotide onone side and the R² or R³ position of the compound is linked to the5′-position of an oligonucleotide on the other side.

In one embodiment, the two different click-carrier compounds comprisecomplementary functional groups and are clicked together to each other.In one embodiment, complementarity functional groups are at R⁵ positionof one click-compound and R² or R³ position of the second compound. Inone embodiment, the complementarity functional groups are at R⁵ positionof one click-compound and R⁵ position of the second compound. In oneembodiment, complementarity functional groups are at R² or R³ positionof one click-compound and R² or R³ position of the second compound.

In some embodiments, B can form part of the click-carrier that connectsthe linker to the carrier. For example, the -linker-Q-linker-R^(L) canbe present at the C2, C6, C7 or C8 position of a purine nucleobase or atthe C2, C5 or C6 position of a pyrimidine nucleobase. The linker can bedirectly attached to the nucleobase or indirectly through one or moreintervening groups such as O, N, S, C(O), C(O)OC(O)NH. In certainembodiments, B, in the click-carrier described above, is uracilyl or auniversal base, e.g., an aryl moiety, e.g., phenyl, optionally havingadditional substituents, e.g., one or more fluoro groups.

In one embodiment, the invention features, a compound having thestructure shown in formula (IV)

wherein R¹⁰ and R²⁰ are independently for each occurrence hydrogen,optionally substituted aliphatic, optionally substituted aryl, oroptionally substituted heteroaryl; R¹, R², R³, R⁴, R⁵, and X are asdefined in the first embodiment.

In one embodiment, the invention features, a compound having thestructure shown in formula (IVa)

wherein T₁ and T₂ are each independently alkyl, C₂-C₉ alkenyl, C₂-C₉alkynyl, substituted C₁-C₉ alkyl, substituted C₁-C₉ alkenyl andsubstituted C₂-C₉ alkynyl; and R1, R2, R3, R4 R5, R10, R20 and X are aspreviously defined.

In one embodiment, the invention features, a compound having thestructure shown in formula (V)

wherein B₁ is halogen, N₃, CN, optionally substituted triazole oroptionally substituted tetrazole. B₁ can also be mesylate. R¹, R², R³,R⁴, and R⁵ are as defined in the first embodiment.

In one embodiment, the invention features, a compound having thestructure shown in formula (Va)

wherein B₁ is halogen, N₃, CN, optionally substituted triazole oroptionally substituted tetrazole; T₁ and T₂ are each independently H,C₁-C₉ alkyl, C₂-C₉ alkenyl, C₂-C₉ alkynyl, substituted C₁-C₉ alkyl,substituted C₁-C₉ alkenyl and substituted C₂-C₉ alkynyl; and R1, R2, R3,R4 and R5 are as previously defined.

In one embodiment, the carrier may be based on the pyrroline ring systemas shown in formula (VI).

wherein E is absent or C(O), C(O)O, C(O)NH, C(S), C(S)NH, SO, SO₂, orSO₂NH;

R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ are each independently foreach occurrence H, —CH₂OR^(a), or OR^(b),

R^(a) and R^(b) are each independently for each occurrence hydrogen,hydroxyl protecting group, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted cycloalkyl, optionallysubstituted aralkyl, optionally substituted alkenyl, optionallysubstituted heteroaryl, polyethyleneglycol (PEG), a phosphate, adiphosphate, a triphosphate, a phosphonate, a phosphonothioate, aphosphonodithioate, a phosphorothioate, a phosphorothiolate, aphosphorodithioate, a phosphorothiolothionate, a phosphodiester, aphosphotriester, an activated phosphate group, an activated phosphitegroup, a phosphoramidite, a solid support, —P(Z¹)(Z²)—O-nucleoside,—P(Z¹)(Z²)—O-oligonucleotide, —P(Z¹)(Z²)-formula (I),—P(Z¹)(O-linker-Q-linker-R^(L))—O-nucleoside,—P(Z¹)(O-linker-N₃)—O-nucleoside, P(Z¹)(O-linker-CN)—O-nucleoside,P(Z¹)(O-linker-C≡R⁸)—O-nucleoside,P(Z¹)(O-linker-cycloalkyne)-O-nucleoside,—P(Z¹)(O-linker-R^(L))—O-oligonucleotide,—P(Z¹)(O-linker-Q-linker-R^(L))—O-oligonucleotide,—P(Z¹)(O-linker-R^(L))—O-oligonucleotide,P(Z¹)(O-linker-N₃)—O-oligonucleotide,—P(Z¹)(O-linker-CN)—O-oligonucleotide,P(Z¹)(O-linker-C≡R⁸)—O-oligonucleotide,P(Z¹)(O-linker-cycloalkyne)-O-oligonucleotide,—P(Z¹)(-linker-Q-linker-R^(L))—O-nucleoside,P(Z¹)(-linker-R^(L))—O-nucleoside, —P(Z¹)(-linker-N₃)—O-nucleoside,P(Z¹)(-linker-CN)—O-nucleoside, P(Z¹)(-linker-C≡R⁸)—O-nucleoside,P(Z¹)(-linker-cycloalkyne)-O-nucleoside,—P(Z¹)(-linker-Q-linker-R^(L))—O-oligonucleotide,(Z¹)(-linker-R^(L))—O-oligonucleotide,P(Z¹)(-linker-N₃)—O-oligonucleotide,—P(Z¹)(-linker-CN)—O-oligonucleotide,P(Z¹)(-linker-C≡R⁸)—O-oligonucleotide orP(Z¹)(-linker-cycloalkyne)-O-oligonucleotide;

R³⁰ is independently for each occurrence -linker-Q-linker-R^(L),-linker-R^(L) or R³¹;

Q is absent or independently for each occurrence

R^(L) is hydrogen or a ligand;

R⁸ is N or CR⁹;

R⁹ is H, optionally substituted alkyl or silyl;

R³¹ is —C(O)CH(N(R³²)₂)(CH₂)_(h)N(R³²)₂;

R³² is independently for each occurrence H, -linker-Q-linker-R^(L),-linker-R^(L) or R³¹;

f and h are independently for each occurrence 1-20; and

Z¹ and Z² are each independently for each occurrence O, S or optionallysubstituted alkyl.

For the pyrroline-based click-carriers, R¹¹ is —CH₂OR^(a) and R³ isOR^(b); or R¹¹ is —CH₂OR^(a) and R⁹ is OR^(b); or R¹¹ is —CH₂OR^(a) andR¹⁷ is OR^(b); or R¹³ is —CH₂OR^(a) and R¹¹ is OR^(b); or R¹³ is—CH₂OR^(a) and R¹⁵ is OR^(b); or R¹³ is —CH₂OR^(a) and R¹⁷ is OR^(b). Incertain embodiments, CH₂OR^(a) and OR^(b) may be geminally substituted.For the 4-hydroxyproline-based carriers, R¹¹ is —CH₂OR^(a) and R¹⁷ isOR^(b). The pyrroline- and 4-hydroxyproline-based compounds maytherefore contain linkages (e.g., carbon-carbon bonds) wherein bondrotation is restricted about that particular linkage, e.g. restrictionresulting from the presence of a ring. Thus, CH₂OR^(a) and OR^(b) may becis or trans with respect to one another in any of the pairingsdelineated above Accordingly, all cis/trans isomers are expresslyincluded. The compounds may also contain one or more asymmetric centersand thus occur as racemates and racemic mixtures, single enantiomers,individual diastereomers and diastereomeric mixtures. All such isomericforms of the compounds are expressly included (e.g., the centers bearingCH₂OR^(a) and OR^(b) can both have the R configuration; or both have theS configuration; or one center can have the R configuration and theother center can have the S configuration and vice versa).

In one embodiment, R¹¹ is CH₂OR^(a) and R⁹ is OR^(b).

In one embodiment, R^(b) is a solid support.

In one embodiment, R³⁰ is —C(O)(CH₂)_(f)NHC(O)(CH₂)_(g)C≡CR⁹; wherein fand g are independently 1-20.

In one embodiment, R³⁹ is

wherein f is 1-20.

In one embodiment, R³⁹ is

wherein f is 1-20.

In one embodiment, R³⁹ is

wherein f is 1-20.

In one embodiment, R³⁹ is

In one embodiment, R³⁹ is

In one preferred embodiment, R³⁹ is R³¹.

In one preferred embodiment, R³¹ is —C(O)CH(N(R³²)₂)(CH₂)₄N(R³²)₂ and atleast one R³² is —C(O)(CH₂)_(f)C≡R⁸ or -linker-Q-linker-R^(L) and R^(L)is present.

In one preferred embodiment, R³¹ is —C(O)CH(N(R³²)₂)(CH₂)₄NH₂ and atleast one R³² is —C(O)(CH₂)_(f)C≡R⁸ or -linker-Q-linker-R^(L) and R^(L)is present.

In one embodiment, R³² is —C(O)(CH₂)₁C≡R⁸.

In one embodiment, R³² is —C(O)(CH₂)₃C≡H.

In one embodiment, R³¹ is —C(O)CH(NH₂)(CH₂)₄NH₂.

In one embodiment features acyclic sugar replacement-based compounds,e.g., sugar replacement based click-carrier compounds, are also referredto herein as ribose replacement compound subunit (RRMS) compoundcompounds. Preferred acyclic carriers can have the structure shown informula (III) or formula (IV) below.

In one aspect, the invention features, an acyclic click-carrier compoundhaving the structure shown in formula (VII)

wherein:

W is absent, O, S and N(R^(N)), where R^(N) is independently for eachoccurrence H, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substituted aryl,optionally substituted cycloalkyl, optionally substituted aralkyl,optionally substituted heteroaryl or an amino protecting group;

E is absent or C(O), C(O)O, C(O)NH, C(S), C(S)NH, SO, SO₂, or SO₂NH;

R^(a) and R^(b) are each independently for each occurrence hydrogen,hydroxyl protecting group, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted cycloalkyl, optionallysubstituted aralkyl, optionally substituted alkenyl, optionallysubstituted heteroaryl, polyethyleneglycol (PEG), a phosphate, adiphosphate, a triphosphate, a phosphonate, a phosphonothioate, aphosphonodithioate, a phosphorothioate, a phosphorothiolate, aphosphorodithioate, a phosphorothiolothionate, a phosphodiester, aphosphotriester, an activated phosphate group, an activated phosphitegroup, a phosphoramidite, a solid support, —P(Z¹)(Z²)—O-nucleoside,—P(Z¹)(Z²)—O-oligonucleotide, —P(Z¹)(Z²)-formula (I),—P(Z¹)(O-linker-Q-linker-R^(L))—O-nucleoside,—P(Z¹)(O-linker-N₃)—O-nucleoside, P(Z¹)(O-linker-CN)—O-nucleoside,P(Z¹)(O-linker-C≡R⁸)—O-nucleoside,P(Z¹)(O-linker-cycloalkyne)-O-nucleoside,—P(Z¹)(O-linker-R^(L))—O-oligonucleotide,—P(Z¹)(O-linker-Q-linker-R^(L))—O-oligonucleotide,—P(Z¹)(O-linker-R^(L))—O-oligonucleotide,P(Z¹)(O-linker-N₃)—O-oligonucleotide,—P(Z¹)(O-linker-CN)—O-oligonucleotide,P(Z¹)(O-linker-C≡R⁸)—O-oligonucleotide,P(Z¹)(O-linker-cycloalkyne)-O-oligonucleotide,—P(Z¹)(-linker-Q-linker-R^(L))—O-nucleoside,P(Z¹)(-linker-R^(L))—O-nucleoside, —P(Z¹)(-linker-N₃)—O-nucleoside,P(Z¹)(-linker-CN)—O-nucleoside, P(Z¹)(-linker-C≡R⁸)—O-nucleoside,P(Z¹)(-linker-cycloalkyne)-O-nucleoside,—P(Z¹)(-linker-Q-linker-R^(L))—O-oligonucleotide,(Z¹)(-linker-R^(L))—O-oligonucleotide,P(Z¹)(-linker-N₃)—O-oligonucleotide,—P(Z¹)(-linker-CN)—O-oligonucleotide,P(Z¹)(-linker-C≡R⁸)—O-oligonucleotide orP(Z¹)(-linker-cycloalkyne)-O-oligonucleotide;

R³⁰ is independently for each occurrence -linker-Q-linker-R^(L),-linker-R^(L) or R³¹;

Q is absent or independently for each occurrence

R^(L) is hydrogen or a ligand;

R⁸ is N or CR⁹

R⁹ is H, optionally substituted alkyl or silyl;

R³¹ is —C(O)CH(N(R³²)₂)(CH₂)_(h)N(R³²)₂;

R³² is independently for each occurrence H, -linker-Q-linker-R^(L) orR³¹;

f and h are independently for each occurrence 1-20;

Z¹ and Z² are each independently for each occurrence O, S or optionallysubstituted alkyl; and

r, s and t are each independently for each occurrence 0, 1, 2 or 3.

When r and s are different, then the tertiary carbon can be either the Ror S configuration. In preferred embodiments, x and y are one and z iszero (e.g. carrier is based on serinol). The acyclic carriers canoptionally be substituted, e.g. with hydroxy, alkoxy, perhaloalky.

In another aspect, the invention features, an acyclic click-carriercompound having the structure shown in formula (VIII)

wherein E is absent or C(O), C(O)O, C(O)NH, C(S), C(S)NH, SO, SO₂, orSO₂NH;

R^(a) and R^(b) are each independently for each occurrence hydrogen,hydroxyl protecting group, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted cycloalkyl, optionallysubstituted aralkyl, optionally substituted alkenyl, optionallysubstituted heteroaryl, polyethyleneglycol (PEG), a phosphate, adiphosphate, a triphosphate, a phosphonate, a phosphonothioate, aphosphonodithioate, a phosphorothioate, a phosphorothiolate, aphosphorodithioate, a phosphorothiolothionate, a phosphodiester, aphosphotriester, an activated phosphate group, an activated phosphitegroup, a phosphoramidite, a solid support, —P(Z¹)(Z²)—O-nucleoside,—P(Z¹)(Z²)—O-oligonucleotide, —P(Z¹)(Z²)-formula (I),—P(Z¹)(O-linker-Q-linker-R^(L))—O-nucleoside,—P(Z¹)(O-linker-N₃)—O-nucleoside, P(Z¹)(O-linker-CN)—O-nucleoside,P(Z¹)(O-linker-C≡R⁸)—O-nucleoside,P(Z¹)(O-linker-cycloalkyne)-O-nucleoside,—P(Z¹)(O-linker-R^(L))—O-oligonucleotide,—P(Z¹)(O-linker-Q-linker-R^(L))—O-oligonucleotide,—P(Z¹)(O-linker-R^(L))—O-oligonucleotide,P(Z¹)(O-linker-N₃)—O-oligonucleotide,—P(Z¹)(O-linker-CN)—O-oligonucleotide,P(Z¹)(O-linker-C≡R⁸)—O-oligonucleotide,P(Z¹)(O-linker-cycloalkyne)-O-oligonucleotide,—P(Z¹)(-linker-Q-linker-R^(L))—O-nucleoside,P(Z¹)(-linker-R^(L))—O-nucleoside, —P(Z¹)(-linker-N₃)—O-nucleoside,P(Z¹)(-linker-CN)—O-nucleoside, P(Z¹)(-linker-C≡R⁸)—O-nucleoside,P(Z¹)(-linker-cycloalkyne)-O-nucleoside,—P(Z¹)(-linker-Q-linker-R^(L))—O-oligonucleotide,(Z¹)(-linker-R^(L))—O-oligonucleotide,P(Z¹)(-linker-N₃)—O-oligonucleotide,—P(Z¹)(-linker-CN)—O-oligonucleotide,P(Z¹)(-linker-C≡R⁸)—O-oligonucleotide orP(Z¹)(-linker-cycloalkyne)-O-oligonucleotide;

R³⁰ is independently for each occurrence -linker-Q-linker-R^(L),-linker-R^(L) or R³¹;

Q is absent or independently for each occurrence

R^(L) is hydrogen or a ligand;

R⁸ is N or CR⁹;

R⁹ is H, optionally substituted alkyl or silyl;

R³¹ is —C(O)CH(N(R³²)₂)(CH₂)_(h)N(R³²)₂;

R³² is independently for each occurrence H, -linker-Q-linker-R^(L) orR³¹;

f and h are independently for each occurrence 1-20;

Z¹ and Z² are each independently for each occurrence O, S or optionallysubstituted alkyl; and

r and s are each independently for each occurrence 0, 1, 2 or 3.

Preferably, Q, —N₃, —CN, or —C≡R⁸ is present at least once in thecompounds of formulas (VI), (VII), and (VIII).

Other carrier compounds amenable to the invention are described incopending applications U.S. Ser. No. 10/916,185, filed Aug. 10, 2004;U.S. Ser. No. 10/946,873, filed Sep. 21, 2004; U.S. Ser. No. 10/985,426,filed Nov. 9, 2004; U.S. Ser. No. 10/833,934, filed Aug. 3, 2007; U.S.Ser. No. 11/115,989 filed Apr. 27, 2005 and U.S. Ser. No. 11/119,533,filed Apr. 29, 2005, which are incorporated by reference in theirentireties for all purposes.

Linkers

The term “linker” means an organic moiety that connects two parts of acompound. Linkers typically comprise a direct bond or an atom such asoxygen or sulfur, a unit such as NR¹, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl, substituted or unsubstituted alkynyl,arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl,heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl,heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl,cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl,alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl,alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl,alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl,alkenylheteroarylalkyl, alkenylheteroarylalkenyl,alkenylheteroarylalkynyl, alkynylheteroarylalkyl,alkynylheteroarylalkenyl, alkynylheteroarylalkynyl,alkylheterocyclylalkyl, alkylheterocyclylalkenyl,alkylhererocyclylalkynyl, alkenylheterocyclylalkyl,alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl,alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl,alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl,alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or moremethylenes can be interrupted or terminated by O, S, S(O), SO₂, N(R¹)₂,C(O), substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocyclic; where R¹ ishydrogen, acyl, aliphatic or substituted aliphatic.

In one embodiment, the linker is represented by structure

—[P-Q₁-R]_(q)-T-,

wherein:

P, R and T are each independently for each occurrence absent, CO, NH, O,S, OC(O), NHC(O), CH₂, CH₂NH, CH₂O; NHCH(R^(a))C(O),—C(O)—CH(R^(a))—NH—, —C(O)-(optionally substituted alkyl)-NH—, CH═N—O,

Q₁ is independently for each occurrence absent, —(CH₂)_(n)—,—C(R¹⁰⁰)(R²⁰⁰)(CH₂)_(n)—, —(CH₂)_(n)C(R¹⁰⁰)(R²⁰⁰)—,—(CH₂CH₂O)_(m)CH₂CH₂—, or —(CH₂CH₂O)_(m)CH₂CH₂NH—;

R^(a) is H or an amino acid side chain;

R¹⁰⁰ and R²⁰⁰ are each independently for each occurrence H, CH₃, OH, SHor N(R^(X))₂;

R^(X) is independently for each occurrence H, methyl, ethyl, propyl,isopropyl, butyl or benzyl;

q is independently for each occurrence 0-20;

n is independently for each occurrence 1-20; and

m is independently for each occurrence 0-50.

In one embodiment, the linker has the structure—[(P-Q₁-R)_(q)—X—(P′-Q₁′-R′)_(q′)]_(q″)-T, wherein:

P, R, T, P′, R′ and T′ are each independently for each occurrenceabsent, CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH, CH₂O; NHCH(R^(a))C(O),—C(O)—CH(R^(a))—NH—, —C(O)-(optionally substituted alkyl)-NH—, CH═N—O,

Q₁ and Q₁′ are each independently for each occurrence absent,—(CH₂)_(n)—, —C(R¹⁰⁰)(R²⁰⁰)(CH₂)_(n)—, —(CH₂)_(n)C(R¹⁰⁰)(R²⁰⁰)—,—(CH₂CH₂O)_(m)CH₂CH₂—, or —(CH₂CH₂O)_(m)CH₂CH₂NH—;

X is a cleavable linking group;

R^(a) is H or an amino acid side chain;

R¹⁰⁰ and R²⁰⁰ are each independently for each occurrence H, CH₃, OH, SHor N(R^(X))₂;

R^(X) is independently for each occurrence H, methyl, ethyl, propyl,isopropyl, butyl or benzyl;

q, q′ and q′ are each independently for each occurrence 0-20;

n is independently for each occurrence 1-20; and

m is independently for each occurrence 0-50.

In one embodiment, the linker comprises at least one cleavable linkinggroup.

Cleavable Linking Groups

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least 10 times or more,preferably at least 100 times faster in the target cell or under a firstreference condition (which can, e.g., be selected to mimic or representintracellular conditions) than in the blood of a subject, or under asecond reference condition (which can, e.g., be selected to mimic orrepresent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some sapcers will have a linkage group that iscleaved at a preferred pH, thereby releasing the iRNA agent from thecarrier oligomer inside the cell, or into the desired compartment of thecell.

A spacer can include a linking group that is cleavable by a particularenzyme. The type of linking group incorporated into a spacer can dependon the cell to be targeted by the iRNA agent. For example, an iRNA agentthat targets an mRNA in liver cells can be linked to the carrieroligomer through a spacer that includes an ester group. Liver cells arerich in esterases, and therefore the tether will be cleaved moreefficiently in liver cells than in cell types that are notesterase-rich. Cleavage of the sapcer releases the iRNA agent from thecarrier oligomer, thereby potentially enhancing silencing activity ofthe iRNA agent. Other cell-types rich in esterases include cells of thelung, renal cortex, and testis.

Spacers that contain peptide bonds can be used when the iRNA agents aretargeting cell types rich in peptidases, such as liver cells andsynoviocytes. For example, an iRNA agent targeted to synoviocytes, suchas for the treatment of an inflammatory disease (e.g., rheumatoidarthritis), can be linked to a carrier oligomer through spacer thatcomprises a peptide bond.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue, e.g., tissue the iRNA agent would be exposed to whenadministered to a subject. Thus one can determine the relativesusceptibility to cleavage between a first and a second condition, wherethe first is selected to be indicative of cleavage in a target cell andthe second is selected to be indicative of cleavage in other tissues orbiological fluids, e.g., blood or serum. The evaluations can be carriedout in cell free systems, in cells, in cell culture, in organ or tissueculture, or in whole animals. It may be useful to make initialevaluations in cell-free or culture conditions and to confirm by furtherevaluations in whole animals. In preferred embodiments, useful candidatecompounds are cleaved at least 2, 4, 10 or 100 times faster in the cell(or under in vitro conditions selected to mimic intracellularconditions) as compared to blood or serum (or under in vitro conditionsselected to mimic extracellular conditions).

Redox Cleavable Linking Groups

One class of cleavable linking groups are redox cleavable linking groupsthat are cleaved upon reduction or oxidation. An example of reductivelycleavable linking group is a disulphide linking group (—S—S—). Todetermine if a candidate cleavable linking group is a suitable“reductively cleavable linking group,” or for example is suitable foruse with a particular iRNA moiety and particular targeting agent one canlook to methods described herein. For example, a candidate can beevaluated by incubation with dithiothreitol (DTT), or other reducingagent using reagents know in the art, which mimic the rate of cleavagewhich would be observed in a cell, e.g., a target cell. The candidatescan also be evaluated under conditions which are selected to mimic bloodor serum conditions. In a preferred embodiment, candidate compounds arecleaved by at most 10% in the blood. In preferred embodiments, usefulcandidate compounds are degraded at least 2, 4, 10 or 100 times fasterin the cell (or under in vitro conditions selected to mimicintracellular conditions) as compared to blood (or under in vitroconditions selected to mimic extracellular conditions). The rate ofcleavage of candidate compounds can be determined using standard enzymekinetics assays under conditions chosen to mimic intracellular media andcompared to conditions chosen to mimic extracellular media.

Phosphate-Based Cleavable Linking Groups

Phosphate-based linking groups are cleaved by agents that degrade orhydrolyze the phosphate group. An example of an agent that cleavesphosphate groups in cells are enzymes such as phosphatases in cells.Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—,—O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—,—S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—,—O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—,—O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—,—O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—,—S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—,—O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—,—O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. Thesecandidates can be evaluated using methods analogous to those describedabove.

Acid Cleavable Linking Groups

Acid cleavable linking groups are linking groups that are cleaved underacidic conditions. In preferred embodiments acid cleavable linkinggroups are cleaved in an acidic environment with a pH of about 6.5 orlower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such asenzymes that can act as a general acid. In a cell, specific low pHorganelles, such as endosomes and lysosomes can provide a cleavingenvironment for acid cleavable linking groups. Examples of acidcleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

Ester-Based Linking Groups

Ester-based linking groups are cleaved by enzymes such as esterases andamidases in cells. Examples of ester-based cleavable linking groupsinclude but are not limited to esters of alkylene, alkenylene andalkynylene groups. Ester cleavable linking groups have the generalformula —C(O)O—, or —OC(O)—. These candidates can be evaluated usingmethods analogous to those described above.

Peptide-Based Cleaving Groups

Peptide-based linking groups are cleaved by enzymes such as peptidasesand proteases in cells. Peptide-based cleavable linking groups arepeptide bonds formed between amino acids to yield oligopeptides (e.g.,dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavablegroups do not include the amide group (—C(O)NH—). The amide group can beformed between any alkylene, alkenylene or alkynelene. A peptide bond isa special type of amide bond formed between amino acids to yieldpeptides and proteins. The peptide based cleavage group is generallylimited to the peptide bond (i.e., the amide bond) formed between aminoacids yielding peptides and proteins and does not include the entireamide functional group. Peptide cleavable linking groups have thegeneral formula —NHCHR¹C(O)NHCHR²C(O)—, where R¹ and R² are the R groupsof the two adjacent amino acids. These candidates can be evaluated usingmethods analogous to those described above.

“Click” Reaction

The synthesis methods of the present invention utilize click chemistryto conjugate the ligand to the click-carrier compound. Click chemistrytechniques are described, for example, in the following references,which are incorporated herein by reference in their entirety:

-   Kolb, H. C.; Finn, M. G. and Sharpless, K. B. Angew. Chem., Int.    Ed. (2001) 40: 2004-2021.-   Kolb, H. C. and Shrapless, K. B. Drug Disc. Today (2003) 8:    112-1137.-   Rostovtsev, V. V.; Green L. G.; Fokin, V. V. and Shrapless, K. B.    Angew. Chem., Int. Ed. (2002) 41: 2596-2599.-   Tornøe, C. W.; Christensen, C. and Meldal, M. J. Org. Chem. (2002)    67: 3057-3064.-   Wang, Q. et al., J. Am. Chem. Soc. (2003) 125: 3192-3193.-   Lee, L. V. et al., J. Am. Chem. Soc. (2003) 125: 9588-9589.-   Lewis, W. G. et al., Angew. Chem., Int. Ed. (2002) 41: 1053-1057.-   Manetsch, R. et al., J. Am. Chem. Soc. (2004) 126: 12809-12818.-   Mocharla, V. P. et al., Angew. Chem., Int. Ed. (2005) 44: 116-120.

Although other click chemistry functional groups can be utilized, suchas those described in the above references, the use of cycloadditionreactions is preferred, particularly the reaction of azides with alkynylgroups. In the presence of Cu(I) salts, terminal alkynes and azidesundergo 1,3-dipolar cycloaddition forming 1,4-disubstituted1,2,3-triazoles. In the presence of Ru(II) salts (e.g. Cu*RuCl(PPh₃)₂),terminal alkynes and azides under go 1,3-dipolar cycloaddition forming1,5-disubstituted 1,2,3-triazoles (Folkin, V. V. et al., Org. Lett.(2005) 127: 15998-15999). Alternatively, a 1,5-disubstituted1,2,3-triazole can be formed using azide and alkynyl reagents (Kraniski,A.; Fokin, V. V. and Sharpless, K. B. Org. Lett. (2004) 6: 1237-1240.Hetero-Diels-Alder reactions or 1,3-dipolar cycloaddition reaction couldalso be used (see for example Padwa, A. 1,3-Dipolar CycloadditionChemistry: Volume 1, John Wiley, New York, (1984) 1-176; Jorgensen, K.A. Angew. Chem., Int. Ed. (2000) 39: 3558-3588 and Tietze, L. F. andKettschau, G. Top. Curr. Chem. (1997) 189: 1-120)

The choice of azides and alkynes as coupling partners is particularlyadvantageous as they are essentially non-reactive towards each other (inthe absence of copper) and are extremely tolerant of other functionalgroups and reaction conditions. This chemical compatibility helps ensurethat many different types of azides and alkynes may be coupled with eachother with a minimal amount of side reactions.

The required copper(I) species are added directly as cuprous salts, forexample CuI, CuOTf.C₆H₆ or [Cu(CH₃CN)₄][PF₆], usually with stabilizingligands (see for example Tornøe, C. W.; Christensen, C. and Meldal, M.J. Org. Chem. (2002) 67: 3057-3064; Chan, T. R. et al., Org. Lett.(2004) 6: 2853-2855; Lewis, W. G. et al., J. Am. Chem. Soc. (2004) 126:9152-9153; Mantovani, G. et al., Chem. Comm. (2005) 2089-2091;Diez-Gonzalez, S. et al., Chem. Eur. J. (2006) 12: 7558-7564 andCandelon, N. et al., Chem. Comm. (2008) 741-743), or more oftengenerated from copper (II) salts with reducing agents (Rostovtsev, V. V.et al., Angew. Chem. (2002) 114: 2708-2711 and Angew. Chem., Int. Ed.(2002) 41: 2596-2599). Metallic copper (for example see Himo, F. et al.,J. Am. Chem. Soc. (2005) 127: 210-216) or clusters (for example seePachon, L. D. et al., Adv. Synth. Catal. (2005) 347: 811-815 andMolteni, G. et al., New J. Chem (2006) 30: 1137-1139) can also beemployed. Chassaing et al., recently reported copper(I) zeolites ascatalysts for the azide-alkyne cycloaddition (Chem. Eur. J. (2008) 14:6713-6721). As copper(I) salts are prone to redox process, nitrogen- orphosphorous-based ligands must be added to protect and stabilize theactive copper catalyst during the cycloaddition reaction.

The reaction is extremely straightforward. The azide and alkyne areusually mixed together in water and a co-solvent such as tert-butanol,THF, DMSO, toluene or DMF. The water/co-solvent are usually in a 1:1 to1:9 ratio. The reactions are usually run overnight although mild heatingshortens reaction times (Sharpless, W. D.; Wu, P.; Hansen, T. V.; andLi, J. G. J. Chem. Ed. (2005) 82: 1833). Aqueous systems can also usecopper(I) species directly such that a reducing agent is not needed. Thereactions conditions then usually require acetonitrile as a co-solvent(although not essential (Chan, T. R.; Hilgraf, R.; Shrapless, K. B. andFokin, V. V. Org. Lett. (2004) 6: 2853)) and a nitrogen base, such astriethylamine, 2,6-lutidine, pyridine and diisopropylamine. In this casecopper(I) species is supplied as CuI, CuOTf.C₆H₆ or [Cu(CH₃CN)₄][PF₆](Rostoctsev, V. V.; Green L. G.; Fokin, V. V. and Shrapless, K. B.Angew. Chem., Int. Ed. (2002) 41: 2596-2599).

Although the water-based methods are attractive for many applications,solvent based azide-alkyne cycloaddition methods have found utility insituations when solubility and/or other problems arise, for example see:

-   Malkoch, M. et al., Macromolecules (2005) 38: 3663.-   Gujadhur, R.; Venkataraman, D. and J. T. Kintigh. J. T. Tet.    Lett. (2001) 42: 4791.-   Laurent, B. A. and Grayson, S. M. J. Am. Chem. Soc. (2006) 128:    4238.-   Opsteen, J. A.; van Hest, J. C. M. Chem. Commun. (2005) 57.-   Tsarevsky, N. V.; Sumerlin, B. S. and Matyjaszewski, K.    Macromolecules (2005) 38: 3558.-   Johnson, J. A. et al., J. Am. Chem. Soc. (2006) 128: 6564.-   Sumerlin, B. S. et al., Macromolecules (2005) 38: 7540.-   Gao, H. F. and Matyjaszewski, K. Macromolecules (2006) 39: 4960.-   Gao, H. et al, Macromolecules (2005) 38: 8979.-   Vogt, A. P. and Sumerlin, B. S. Macromolecules (2006) 39: 5286.-   Lutz, J. F.; Borner, H. G. and Weichenhan, K. Macromol. Rapid    Comm. (2005) 26: 514.-   Mantovani, G.; Ladmiral, V.; Tao, L. and Haddleton, D. M. Chem.    Comm. (2005) 2089.

The click reaction may be performed thermally. In one aspect, the clickreaction is performed at slightly elevated temperatures between 25° C.and 100° C. In one aspect, the reaction may be performed between 25° C.and 75° C., or between 25° C. and 65° C., or between 25° C. and 50° C.In one embodiment, the reaction is performed at room temperature. Inanother aspect, the click reaction may also be performed using amicrowave oven. The microwave assisted click reaction may be carried outin the presence or absence of copper.

In one aspect, the invention provides a method for coupling aclick-carrier compound to a ligand through a click reaction. In apreferred embodiment, the click reaction is a cycloaddition reaction ofazide with alkynyl group and catalyzed by copper. In one embodiment theequal molar amount of alkyne and azide are mixed together in DCM/MeOH(10:1 to 1:1 ratio v/v) and 0.05-0.5 mol % each of [Cu(CH₃CN)₄][PF₆] andcopper are added the reaction. In one embodiment DCM/MeOH ratio is 5:1to 1:1. In a preferred embodiment, DCM/MeOH ratio is 4:1. In oneembodiment, equal molar amounts of [Cu(CH₃CN)₄][PF₆] and copper areadded. In a preferred embodiment, 0.05-0.25 mol % each of[Cu(CH₃CN)₄][PF₆] and copper are added to the reaction. In a morepreferred embodiment, 0.05 mol %, 0.1 mol %, 0.15 mol %, 0.2 mol % or0.25 mol % each of [Cu(CH₃CN)₄][PF₆] and copper are added to thereaction.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular, prodrug versions of theoligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl)phosphate]derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the oligomeric compounds of theinvention: i.e., salts that retain the desired biological activity ofthe parent compound and do not impart undesired toxicological effectsthereto. For oligonucleotides, preferred examples of pharmaceuticallyacceptable salts and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety.

Ligands

A wide variety of entities can be coupled to the oligonucleotide, e.g.the iRNA agent, using the “click” reaction. Preferred entities can becoupled to the oligonucleotide at various places, for example, 3′-end,5′-end, and/or at internal positions.

In preferred embodiments, the ligand is attached to the iRNA agent viaan intervening linker. The ligand may be present on a compound when saidcompound is incorporated into the growing strand. In some embodiments,the ligand may be incorporated via coupling to a “precursor” compoundafter said “precursor” compound has been incorporated into the growingstrand. For example, a compound having, e.g., an azide terminated linker(i.e., having no associated ligand), e.g., -linker-N₃ may beincorporated into a growing sense or antisense strand. In a subsequentoperation, i.e., after incorporation of the precursor compound into thestrand, a ligand having an alkyne, e.g. terminal acetylene, e.g.ligand-C≡CH, can subsequently be attached to the precursor compound bythe “click” reaction. Alternatively, the compound linker comprises analkyne, e.g. terminal acetylene; and the ligand comprises an azidefunctionality for the “click” reaction to take place. The azide oralkyne functionalities can be incorporated into the ligand by methodsknown in the art. For example, moieties carrying azide or alkynefunctionalities can be linked to the ligand or a functional group on theligand can be transformed into an azide or alkyne. In one embodiment,the conjugation of the ligand to the precursor compound takes placewhile the oligonucleotide is still attached to the solid support. In oneembodiment, the precursor carrying oligonucleotide is first deprotectedbut not purified before the ligand conjugation takes place. In oneembodiment, the precursor compound carrying oligonucleotide is firstdeprotected and purified before the ligand conjugation takes place. Incertain embodiments, the “click” reaction is carried out undermicrowave.

In preferred embodiments, a ligand alters the distribution, targeting orlifetime of an iRNA agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand. Preferred ligands will nottake part in duplex pairing in a duplexed nucleic acid.

Preferred ligands can have endosomolytic properties. The endosomolyticligands promote the lysis of the endosome and/or transport of thecomposition of the invention, or its components, from the endosome tothe cytoplasm of the cell. The endosomolytic ligand may be a polyanionicpeptide or peptidomimetic which shows pH-dependent membrane activity andfusogenicity. In certain embodiments, the endosomolytic ligand assumesits active conformation at endosomal pH. The “active” conformation isthat conformation in which the endosomolytic ligand promotes lysis ofthe endosome and/or transport of the composition of the invention, orits components, from the endosome to the cytoplasm of the cell.Exemplary endosomolytic ligands include the GALA peptide (Subbarao etal., Biochemistry, 1987, 26: 2964-2972), the EALA peptide (Vogel et al.,J. Am. Chem. Soc., 1996, 118: 1581-1586), and their derivatives (Turk etal., Biochem. Biophys. Acta, 2002, 1559: 56-68). In certain embodiments,the endosomolytic component may contain a chemical group (e.g., an aminoacid) which will undergo a change in charge or protonation in responseto a change in pH. The endosomolytic component may be linear orbranched. Exemplary primary sequences of peptide based endosomolyticligands are shown in table 1.

TABLE 1 List of peptides with endosomolytic activity. NameSequence (N to C) Ref. GALA AALEALAEALEALAEALEALAEAAAAGGC 1 EALAAALAEALAEALAEALAEALAEALAAAAGGC 2 ALEALAEALEALAEA 3 INF-7GLFEAIEGFIENGWEGMIWDYG 4 Inf HA-2 GLFGAIAGFIENGWEGMIDGWYG 5 diINF-7GLF EAI EGFI ENGW EGMI DGWYGC 5 GLF EAI EGFI ENGW EGMI DGWYGC diINF3GLF EAI EGFI ENGW EGMI DGGC 6 GLF EAI EGFI ENGW EGMI DGGC GLFGLFGALAEALAEALAEHLAEALAEALEALAAGG 6 SC GALA-INF3GLFEAIEGFIENGWEGLAEALAEALEALAAGGSC 6 INF-5 GLF EAI EGFI ENGW EGnI DG K 4GLF EAI EGFI ENGW EGnI DG n, norleucine

REFERENCES

-   1. Subbarao et al. (1987) Biochemistry 26: 2964-2972.-   2. Vogel, et al. (1996) J. Am. Chem. Soc. 118: 1581-1586-   3. Turk, et al. (2002) Biochim. Biophys. Acta 1559: 56-68.-   4. Plank, et al. (1994) J. Biol. Chem. 269:12918-12924.-   5. Mastrobattista, et al. (2002) J. Biol. Chem. 277:27135-43.-   6. Oberhauser, et al. (1995) Deliv. Strategies Antisense    Oligonucleotide Ther. 247-66.

Preferred ligands can improve transport, hybridization, and specificityproperties and may also improve nuclease resistance of the resultantnatural or modified oligoribonucleotide, or a polymeric moleculecomprising any combination of compounds described herein and/or naturalor modified ribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., forenhancing uptake; diagnostic compounds or reporter groups e.g., formonitoring distribution; cross-linking agents; and nuclease-resistanceconferring moieties. General examples include lipids, steroids,vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL),high-density lipoprotein (HDL), or globulin); an carbohydrate (e.g., adextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronicacid); or a lipid. The ligand may also be a recombinant or syntheticmolecule, such as a synthetic polymer, e.g., a synthetic polyamino acid,an oligonucleotide (e.g. an aptamer). Examples of polyamino acidsinclude polyamino acid is a polylysine (PLL), poly L-aspartic acid, polyL-glutamic acid, styrene-maleic acid anhydride copolymer,poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydridecopolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGDpeptide mimetic or an aptamer. Table 2 shows some examples of targetingligands and their associated receptors.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g, cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

TABLE 2 Liver targeting Ligands and their associated receptors LiverCells Ligand Receptor 1) Parenchymal Galactose ASGP-R Cell (PC)(Asiologlycoprotein (Hepatocytes) receptor) Gal NAc ASPG-R (GalNAc(n-acetyl-galactosamine) Receptor) Lactose Asialofetuin ASPG-r 2)Sinusoidal Hyaluronan Hyaluronan receptor Endothelial Cell ProcollagenProcollagen receptor (SEC) Negatively charged Scavenger receptorsmolecules Mannose Mannose receptors N-acetyl Glucosamine Scavengerreceptors Immunoglobulins Fc Receptor LPS CD14 Receptor Insulin Receptormediated transcytosis Transferrin Receptor mediated transcytosisAlbumins Non-specific Sugar-Albumin conjugates Mannose-6-phosphateMannose-6-phosphate receptor 3) Kupffer Cell Mannose Mannose receptors(KC) Fucose Fucose receptors Albumins Non-specific Mannose-albuminconjugates

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell. Ligands may also include hormonesand hormone receptors. They can also include non-peptidic species, suchas lipids, lectins, carbohydrates, vitamins, cofactors, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose, oraptamers. The ligand can be, for example, a lipopolysaccharide, anactivator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g, a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

The ligand can increase the uptake of the iRNA agent into the cell byactivating an inflammatory response, for example. Exemplary ligands thatwould have such an effect include tumor necrosis factor alpha(TNFalpha), interleukin-1 beta, or gamma interferon.

In one aspect, the ligand is a lipid or lipid-based molecule. Such alipid or lipid-based molecule preferably binds a serum protein, e.g.,human serum albumin (HSA). An HSA binding ligand allows for distributionof the conjugate to a target tissue, e.g., a non-kidney target tissue ofthe body. For example, the target tissue can be the liver, includingparenchymal cells of the liver. Other molecules that can bind HSA canalso be used as ligands. For example, neproxin or aspirin can be used. Alipid or lipid-based ligand can (a) increase resistance to degradationof the conjugate, (b) increase targeting or transport into a target cellor cell membrane, and/or (c) can be used to adjust binding to a serumprotein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells. Also included are HAS, low density lipoprotein (LDL) andhigh-density lipoprotein (HDL).

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to iRNA agentscan affect pharmacokinetic distribution of the iRNA, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long (see Table 3, forexample).

TABLE 3 Exemplary Cell Permeation Peptides Cell Permeation PeptideAmino acid Sequence Reference Penetratin RQIKIWFQNRRMKWKKDerossi et al., J. Biol. Chem. 269: 10444, 1994 Tat fragmentGRKKRRQRRRPPQC Vives et al., J. (48-60) Biol. Chem., 272: 16010, 1997Signal GALFLGWLGAAGSTMGAWSQPKKKRKV Chaloin et al., Sequence- Biochem.based peptide Biophys. Res. Commun., 243: 601, 1998 PVECLLIILRRRIRKQAHAHSK Elmquist et al., Exp. Cell Res., 269: 237, 2001Transportan GWTLNSAGYLLKINLKALAALAKKIL Pooga et al., FASEB J.,12: 67, 1998 Amphiphilic KLALKLALKALKAALKLA Oehlke et al., model peptideMol. Ther., 2: 339, 2000 Arg₉ RRRRRRRRR Mitchell et al., J. Pept. Res.,56: 318, 2000 Bacterial cell KFFKFFKFFK wall permeating LL-37LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNL VPRTES Cecropin P1SWLSKTAKKLENSAKKRISEGIAIAIQGGPR α-defensinACYCRIPACIAGERRYGTCIYQGRLWAFCC b-defensin DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK ) Bactenecin RKCRIVVIRVCR PR-39 RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFPGKR-NH2 Indolicidin ILPWKWPWWPWRR-NH2

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP. An RFGF analogue (e.g., amino acid sequenceAALLPVLLAAP) containing a hydrophobic MTS can also be a targetingmoiety. The peptide moiety can be a “delivery” peptide, which can carrylarge polar molecules including peptides, oligonucleotides, and proteinacross cell membranes. For example, sequences from the HIV Tat protein(GRKKRRQRRRPPQ) and the Drosophila Antennapedia protein(RQIKIWFQNRRMKWKK) have been found to be capable of functioning asdelivery peptides. A peptide or peptidomimetic can be encoded by arandom sequence of DNA, such as a peptide identified from aphage-display library, or one-bead-one-compound (OBOC) combinatoriallibrary (Lam et al., Nature, 354:82-84, 1991). Preferably the peptide orpeptidomimetic tethered to an iRNA agent via an incorporated compoundunit is a cell targeting peptide such as an arginine-glycine-asparticacid (RGD)-peptide, or RGD mimic. A peptide moiety can range in lengthfrom about 5 amino acids to about 40 amino acids. The peptide moietiescan have a structural modification, such as to increase stability ordirect conformational properties. Any of the structural modificationsdescribed below can be utilized.

An RGD peptide moiety can be used to target a tumor cell, such as anendothelial tumor cell or a breast cancer tumor cell (Zitzmann et al.,Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targetingof an iRNA agent to tumors of a variety of other tissues, including thelung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy8:783-787, 2001). Preferably, the RGD peptide will facilitate targetingof an iRNA agent to the kidney. The RGD peptide can be linear or cyclic,and can be modified, e.g., glycosylated or methylated to facilitatetargeting to specific tissues. For example, a glycosylated RGD peptidecan deliver an iRNA agent to a tumor cell expressing α_(v)β₃ (Haubner etal., Jour. Nucl. Med., 42:326-336, 2001).

Peptides that target markers enriched in proliferating cells can beused. E.g., RGD containing peptides and peptidomimetics can targetcancer cells, in particular cells that exhibit an I_(v)θ₃ integrin.Thus, one could use RGD peptides, cyclic peptides containing RGD, RGDpeptides that include D-amino acids, as well as synthetic RGD mimics. Inaddition to RGD, one can use other moieties that target the I_(v)-θ₃integrin ligand. Generally, such ligands can be used to controlproliferating cells and angiogeneis. Preferred conjugates of this typeligands that targets PECAM-1, VEGF, or other cancer gene, e.g., a cancergene described herein.

TABLE 4 Azide modified peptides. NB12675N3-(CH2)5-CO-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln-NH2 NB12707N3-(CH2)15-CO-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln-NH2 NB12676cyclo-[Phe-Arg-Gly-Asp-Lys(N3-(CH2)5- COO H )] NB12708cyclo-[Phe-Arg-Gly-Asp-Lys(N3-(CH2)15- COOH)] NB12709N3-(CH2)5-CO-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Arg-Arg-NH2 NB12710N3-(CH2)15-CO-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Arg-Arg-NH2

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., a -defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

In one embodiment, a targeting peptide tethered to an iRNA agent and/orthe carrier oligomer can be an amphipathic α-helical peptide. Exemplaryamphipathic α-helical peptides include, but are not limited to,cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide(BLP), cathelicidins, ceratotoxins, S. clava peptides, hagfishintestinal antimicrobial peptides (HFIAPs), magainines, brevinins-2,dermaseptins, melittins, pleurocidin, H₂A peptides, Xenopus peptides,esculentinis-1, and caerins. A number of factors will preferably beconsidered to maintain the integrity of helix stability. For example, amaximum number of helix stabilization residues will be utilized (e.g.,leu, ala, or lys), and a minimum number helix destabilization residueswill be utilized (e.g., proline, or cyclic compoundic units. The cappingresidue will be considered (for example Gly is an exemplary N-cappingresidue and/or C-terminal amidation can be used to provide an extraH-bond to stabilize the helix. Formation of salt bridges betweenresidues with opposite charges, separated by i±3, or i±4 positions canprovide stability. For example, cationic residues such as lysine,arginine, homo-arginine, ornithine or histidine can form salt bridgeswith the anionic residues glutamate or aspartate.

Peptide and peptidomimetic ligands include those having naturallyoccurring or modified peptides, e.g., D or L peptides; α, β, or γpeptides; N-methyl peptides; azapeptides; peptides having one or moreamide, i.e., peptide, linkages replaced with one or more urea, thiourea,carbamate, or sulfonyl urea linkages; or cyclic peptides.

The targeting ligand can be any ligand that is capable of targeting aspecific receptor. Examples are: folate, GalNAc, GalNAc₃, galactose,mannose, mannose-6P, clusters of sugars such as GalNAc cluster, mannosecluster, galactose cluster, or an aptamer. A cluster is a combination oftwo or more sugar units. The targeting ligands also include integrinreceptor ligands, Chemokine receptor ligands, transferrin, biotin,serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDLand HDL ligands. The ligands can also be based on nucleic acid, e.g., anaptamer. The aptamer can be unmodified or have any combination ofmodifications disclosed herein.

Endosomal release agents include imidazoles, poly or oligoimidazoles,PEIs, peptides, fusogenic peptides, polycaboxylates, polyacations,masked oligo or poly cations or anions, acetals, polyacetals,ketals/polyketyals, orthoesters, polymers with masked or unmaskedcationic or anionic charges, dendrimers with masked or unmasked cationicor anionic charges.

PK modulator stands for pharmacokinetic modulator. PK modulator includelipophiles, bile acids, steroids, phospholipid analogues, peptides,protein binding agents, PEG, vitamins etc. Examplary PK modulatorinclude, but are not limited to, cholesterol, fatty acids, cholic acid,lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids,sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc.Oligonucleotides that comprise a number of phosphorothioate linkages arealso known to bind to serum protein, thus short oligonucleotides, e.g.oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases,comprising multiple of phosphorothioate linkages in the backbaone arealso amenable to the present invention as ligands (e.g. as PK modulatingligands).

In addition, aptamers that bind serum components (e.g. serum proteins)are also amenable to the present invention as PK modulating ligands.

Other ligands amenable to the invention are described in copendingapplications U.S. Ser. No. 10/916,185, filed Aug. 10, 2004; U.S. Ser.No. 10/946,873, filed Sep. 21, 2004; U.S. Ser. No. 10/833,934, filedAug. 3, 2007; U.S. Ser. No. 11/115,989 filed Apr. 27, 2005 and U.S. Ser.No. 11/944,227 filed Nov. 21, 2007, which are incorporated by referencein their entireties for all purposes.

When two or more ligands are present, the ligands can all have sameproperties, all have different properties or some ligands have the sameproperties while others have different properties. For example, a ligandcan have targeting properties, have endosomolytic activity or have PKmodulating properties. In a preferred embodiment, all the ligands havedifferent properties.

The compound comprising the ligand, e.g. the click-carrier compound, canbe present in any position of an oligonucleotide, e.g. an iRNA agent. Insome embodiments, click-carrier compound can be present at the terminussuch as a 5′ or 3′ terminal of the iRNA agent. Click-carrier compoundscan also present at an internal position of the iRNA agent. Fordouble-stranded iRNA agents, click-carrier compounds can be incorporatedinto one or both strands. In some embodiments, the sense strand of thedouble-stranded iRNA agent comprises the click-carrier compound. Inother embodiments, the antisense strand of the double-stranded iRNAagent comprises the click-carrier compound.

In some embodiments, ligands can be conjugated to nucleobases, sugarmoieties, or internucleosidic linkages of nucleic acid molecules.Conjugation to purine nucleobases or derivatives thereof can occur atany position including, endocyclic and exocyclic atoms. In someembodiments, the 2-, 6-, 7-, or 8-positions of a purine nucleobase areattached to a conjugate moiety. Conjugation to pyrimidine nucleobases orderivatives thereof can also occur at any position. In some embodiments,the 2-, 5-, and 6-positions of a pyrimidine nucleobase can besubstituted with a conjugate moiety. Conjugation to sugar moieties ofnucleosides can occur at any carbon atom. Example carbon atoms of asugar moiety that can be attached to a conjugate moiety include the 2′,3′, and 5′ carbon atoms. The 1′ position can also be attached to aconjugate moiety, such as in an abasic residue. Internucleosidiclinkages can also bear conjugate moieties. For phosphorus-containinglinkages (e.g., phosphodiester, phosphorothioate, phosphorodithiotate,phosphoroamidate, and the like), the conjugate moiety can be attacheddirectly to the phosphorus atom or to an O, N, or S atom bound to thephosphorus atom. For amine- or amide-containing internucleosidiclinkages (e.g., PNA), the conjugate moiety can be attached to thenitrogen atom of the amine or amide or to an adjacent carbon atom.

There are numerous methods for preparing conjugates of oligomericcompounds. Generally, an oligomeric compound is attached to a conjugatemoiety by contacting a reactive group (e.g., OH, SH, amine, carboxyl,aldehyde, and the like) on the oligomeric compound with a reactive groupon the conjugate moiety. In some embodiments, one reactive group iselectrophilic and the other is nucleophilic.

For example, an electrophilic group can be a carbonyl-containingfunctionality and a nucleophilic group can be an amine or thiol. Methodsfor conjugation of nucleic acids and related oligomeric compounds withand without linking groups are well described in the literature such as,for example, in Manoharan in Antisense Research and Applications, Crookeand LeBleu, eds., CRC Press, Boca Raton, Fla., 1993, Chapter 17, whichis incorporated herein by reference in its entirety.

Representative United States patents that teach the preparation ofoligonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,149,782; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; 5,672,662;5,688,941; 5,714,166; 6,153,737; 6,172,208; 6,300,319; 6,335,434;6,335,437; 6,395,437; 6,444,806; 6,486,308; 6,525,031; 6,528,631;6,559,279; each of which is herein incorporated by reference.

Oligonucleotide

The term “oligonucleotide” as used herein refers to an unmodified RNA,modified RNA, or nucleoside surrogate, all of which are defined herein.Although the modifications are described in context of an iRNA agent, itis understood that these modifications are also applicable to otheroligonucleotides of the invention such as antisense, antagomir, aptamer,ribozyme and decoy oligonucleotides. While numerous modified RNAs andnucleoside surrogates are described, preferred examples include thosewhich have greater resistance to nuclease degradation than do unmodifiedRNAs. Preferred examples include those which have a 2′ sugarmodification, a modification in a single strand overhang, preferably a3′ single strand overhang, or, particularly if single stranded, a 5′modification which includes one or more phosphate groups or one or moreanalogs of a phosphate group.

An “iRNA agent” as used herein, is an RNA agent which can, or which canbe cleaved into an RNA agent which can, down regulate the expression ofa target gene, preferably an endogenous or pathogen target RNA. Whilenot wishing to be bound by theory, an iRNA agent may act by one or moreof a number of mechanisms, including post-transcriptional cleavage of atarget mRNA sometimes referred to in the art as RNAi, orpre-transcriptional or pre-translational mechanisms. An iRNA agent caninclude a single strand or can include more than one strands, e.g., itcan be a double stranded iRNA agent. If the iRNA agent is a singlestrand it is particularly preferred that it include a 5′ modificationwhich includes one or more phosphate groups or one or more analogs of aphosphate group. If the iRNA agent is double stranded the doublestranded region can include more than two or more strands, e.g, twostrands, e.g. three strands, in the double stranded region.

The iRNA agent should include a region of sufficient homology to thetarget gene, and be of sufficient length in terms of nucleotides, suchthat the iRNA agent, or a fragment thereof, can mediate down regulationof the target gene. (For ease of exposition the term nucleotide orribonucleotide is sometimes used herein in reference to one or morecompoundic subunits of an RNA agent. It will be understood herein thatthe usage of the term “ribonucleotide” or “nucleotide”, herein can, inthe case of a modified RNA or nucleotide surrogate, also refer to amodified nucleotide, or surrogate replacement moiety at one or morepositions.) Thus, the iRNA agent is or includes a region which is atleast partially, and in some embodiments fully, complementary to thetarget RNA. It is not necessary that there be perfect complementaritybetween the iRNA agent and the target, but the correspondence must besufficient to enable the iRNA agent, or a cleavage product thereof, todirect sequence specific silencing, e.g., by RNAi cleavage of the targetRNA, e.g., mRNA.

An iRNA agent will often be modified or include nucleoside surrogates inaddition to the click-carrier compound. Single stranded regions of aniRNA agent will often be modified or include nucleoside surrogates,e.g., the unpaired region or regions of a hairpin structure, e.g., aregion which links two complementary regions, can have modifications ornucleoside surrogates. Modification to stabilize one or more 3′- or5′-terminus of an iRNA agent, e.g., against exonucleases, or to favorthe antisense sRNA agent to enter into RISC are also favored.Modifications can include C3 (or C6, C7, C12) amino linkers, thiollinkers, carboxyl linkers, non-nucleotidic spacers (C3, C6, C9, C12,abasic, triethylene glycol, hexaethylene glycol), special biotin orfluorescein reagents that come as phosphoramidites and that have anotherDMT-protected hydroxyl group, allowing multiple couplings during RNAsynthesis.

A “single strand iRNA agent” as used herein, is an iRNA agent which ismade up of a single molecule. It may include a duplexed region, formedby intra-strand pairing, e.g., it may be, or include, a hairpin orpan-handle structure. Single strand iRNA agents are preferably antisensewith regard to the target molecule. In preferred embodiments singlestrand iRNA agents are 5′-phosphorylated or include a phosphoryl analogat the 5′-terminus. 5′-phosphate modifications include those which arecompatible with RISC mediated gene silencing. Suitable modificationsinclude: 5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylatedor non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′);5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination ofoxygen/sulfur replaced monophosphate, diphosphate and triphosphates(e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-). (These modifications can also be used with theantisense strand of a multi-strand iRNA agent.)

A “multi-strand iRNA agent” as used herein, is an iRNA agent whichcomprises two or more strands, for example a double-stranded iRNA agent.The strands form duplexed regions and may include a hairpin, pan-handlestructure, loop or bulges. At least one strand of the iRNA agent ispreferably antisense with regard to the target molecule.

It may be desirable to modify only one, only two or all strands of amulti-strand iRNA agent. In some cases they will have the samemodification or the same class of modification but in other cases thedifferent strand will have different modifications, e.g., in some casesit is desirable to modify only one strand. It may be desirable to modifyonly some strands, e.g., to inactivate them, e.g., strands can bemodified in order to inactivate them and prevent formation of an activeiRNA/protein or RISC. This can be accomplished by a modification whichprevents 5′-phosphorylation of the strands, e.g., by modification with a5′-O-methyl ribonucleotide (see Nykänen et al., (2001) ATP requirementsand small interfering RNA structure in the RNA interference pathway.Cell 107, 309-321.) Other modifications which prevent phosphorylationcan also be used, e.g., simply substituting the 5′-OH by H rather than0-Me. Alternatively, a large bulky group may be added to the5′-phosphate turning it into a phosphodiester linkage, though this maybe less desirable as phosphodiesterases can cleave such a linkage andrelease a functional iRNA 5′-end. Antisense strand modifications include5′-phosphorylation as well as any of the other 5′ modificationsdiscussed herein, particularly the 5′ modifications discussed above inthe section on single stranded iRNA molecules.

In some cases, the different strands will include differentmodifications. Multiple different modifications can be included on eachof the strands. The modifications on a given strand may differ from eachother, and may also differ from the various modifications on otherstrands. For example, one strand may have a modification, e.g., amodification described herein, and a different strand may have adifferent modification, e.g., a different modification described herein.In other cases, one strand may have two or more different modifications,and the another strand may include a modification that differs from theat least two modifications on the other strand.

It is preferred that the strands be chosen such that the iRNA agentincludes a single strand or unpaired region at one or both ends of themolecule. Thus, an iRNA agent contains two or more strands, preferablepaired to contain an overhang, e.g., one or two 5′ or 3′ overhangs butpreferably a 3′ overhang of 2-3 nucleotides. Most embodiments will havea 3′ overhang. Preferred iRNA agents will have single-strandedoverhangs, preferably 3′ overhangs, of 1 or preferably 2 or 3nucleotides in length at each end. The overhangs can be the result ofone strand being longer than the other, or the result of two strands ofthe same length being staggered.

Preferred lengths for the duplexed regions between the strands arebetween 6 and 30 nucleotides in length. The preferred duplexed regionsare between 15 and 30, most preferably 18, 19, 20, 21, 22, and 23nucleotides in length. Other preferred duplexed regions are between 6and 20 nucleotides, most preferably 6, 7, 8, 9, 10, 11 and 12nucleotides in length. In multi-strand iRNA agents different duplexesformed may have different lengths, e.g. duplexed region formed betweenstrand A and B may have a different length than duplexed region formedbetween strand A and C.

In iRNA agents comprising more than two strands duplexed agents canresemble in length and structure the natural Dicer processed productsfrom long dsRNAs. Embodiments in which the two or more strands of theiRNA agent are linked, e.g., covalently linked are also included.Hairpins or other single strand structures which provide the requireddouble stranded region, and preferably a 3′ overhang are also within theinvention.

As nucleic acids are polymers of subunits or compounds, many of themodifications described below occur at a position which is repeatedwithin a nucleic acid, e.g., a modification of a base, or a phosphatemoiety, or the non-bridging oxygen of a phosphate moiety. In some casesthe modification will occur at all of the subject positions in thenucleic acid but in many, and in fact in most cases it will not. By wayof example, a modification may only occur at a 3′ or 5′ terminalposition, may only occur in the internal unpaired region, may only occurin a terminal regions, e.g. at a position on a terminal nucleotide or inthe last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification mayoccur in a double strand region, a single strand region, or in both. Amodification may occur only in the double strand region of an RNA agentor may only occur in a single strand region of an RNA agent. E.g., aphosphorothioate modification at a non-bridging oxygen position may onlyoccur at one or both termini, may only occur in a terminal regions,e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5,or 10 nucleotides of a strand, or may occur in double strand and singlestrand regions, particularly at termini. The 5′ end or ends can bephosphorylated.

In some embodiments it is particularly preferred, e.g., to enhancestability, to include particular bases in overhangs, or to includemodified nucleotides or nucleotide surrogates, in single strandoverhangs, e.g., in a 5′ or 3′ overhang, or in both. E.g., it can bedesirable to include purine nucleotides in overhangs. In someembodiments all or some of the bases in a 3′ or 5′ overhang will bemodified, e.g., with a modification described herein. Modifications caninclude, e.g., the use of modifications at the 2′ OH group of the ribosesugar, e.g., the use of deoxyribonucleotides, e.g., deoxythymidine,instead of ribonucleotides, and modifications in the phosphate group,e.g., phosphothioate modifications. Overhangs need not be homologouswith the target sequence.

MicroRNAs

MicroRNAs (miRNAs or mirs) are a highly conserved class of small RNAmolecules that are transcribed from DNA in the genomes of plants andanimals, but are not translated into protein. Pre-microRNAs areprocessed into miRNAs. Processed microRNAs are single stranded ˜17-25nucleotide (nt) RNA molecules that become incorporated into theRNA-induced silencing complex (RISC) and have been identified as keyregulators of development, cell proliferation, apoptosis anddifferentiation. They are believed to play a role in regulation of geneexpression by binding to the 3′-untranslated region of specific mRNAs.RISC mediates down-regulation of gene expression through translationalinhibition, transcript cleavage, or both. RISC is also implicated intranscriptional silencing in the nucleus of a wide range of eukaryotes.

MicroRNAs have also been implicated in modulation of pathogens in hosts.For example, see Jopling, C. L., et al., Science (2005) vol. 309, pp1577-1581. Without wishing to be bound by theory, administration of amicroRNA, microRNA mimic, and/or anti microRNA oligonucleotide, leads tomodulation of pathogen viability, growth, development, and/orreplication. In certain embodiments, the oligonucleotide is a microRNA,microRNA mimic, and/or anti microRNA, wherein microRNA is a hostmicroRNA.

The number of miRNA sequences identified to date is large and growing,illustrative examples of which can be found, for example, in: “miRBase:microRNA sequences, targets and gene nomenclature” Griffiths-Jones S,Grocock R J, van Dongen S, Bateman A, Enright A J. NAR, 2006, 34,Database Issue, D140-D144; “The microRNA Registry” Griffiths-Jones S.NAR, 2004, 32, Database Issue, D109-D111; and also on the worldwide webat http://microrna.dot.sanger.dot.ac.dot.uk/sequences/.

Ribozymes

Ribozymes are oligonucleotides having specific catalytic domains thatpossess endonuclease activity (Kim and Cech, Proc Natl Acad Sci USA.1987 December; 84(24):8788-92; Forster and Symons, Cell. 1987 Apr. 24;49(2):211-20). At least six basic varieties of naturally-occurringenzymatic RNAs are known presently. In general, enzymatic nucleic acidsact by first binding to a target RNA. Such binding occurs through thetarget binding portion of an enzymatic nucleic acid which is held inclose proximity to an enzymatic portion of the molecule that acts tocleave the target RNA. Thus, the enzymatic nucleic acid first recognizesand then binds a target RNA through complementary base-pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its ability todirect synthesis of an encoded protein. After an enzymatic nucleic acidhas bound and cleaved its RNA target, it is released from that RNA tosearch for another target and can repeatedly bind and cleave newtargets.

Methods of producing a ribozyme targeted to any target sequence areknown in the art. Ribozymes can be designed as described in Int. Pat.Appl. Publ. No. WO 93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595,each specifically incorporated herein by reference, and synthesized tobe tested in vitro and in vivo, as described therein.

Aptamers

Aptamers are nucleic acid or peptide molecules that bind to a particularmolecule of interest with high affinity and specificity (Tuerk and Gold,Science 249:505 (1990); Ellington and Szostak, Nature 346:818 (1990)).DNA or RNA aptamers have been successfully produced which bind manydifferent entities from large proteins to small organic molecules. SeeEaton, Curr. Opin. Chem. Biol. 1:10-16 (1997), Famulok, Curr. Opin.Struct. Biol. 9:324-9(1999), and Hermann and Patel, Science 287:820-5(2000). Aptamers can be RNA or DNA based. Generally, aptamers areengineered through repeated rounds of in vitro selection orequivalently, SELEX (systematic evolution of ligands by exponentialenrichment) to bind to various molecular targets such as smallmolecules, proteins, nucleic acids, and even cells, tissues andorganisms. The aptamer can be prepared by any known method, includingsynthetic, recombinant, and purification methods, and can be used aloneor in combination with other aptamers specific for the same target.Further, as described more fully herein, the term “aptamer” specificallyincludes “secondary aptamers” containing a consensus sequence derivedfrom comparing two or more known aptamers to a given target.

Decoy Oligonucleotides

Because transcription factors recognize their relatively short bindingsequences, even in the absence of surrounding genomic DNA, shortoligonucleotides bearing the consensus binding sequence of a specifictranscription factor can be used as tools for manipulating geneexpression in living cells. This strategy involves the intracellulardelivery of such “decoy oligonucleotides”, which are then recognized andbound by the target factor. Occupation of the transcription factor'sDNA-binding site by the decoy renders the transcription factor incapableof subsequently binding to the promoter regions of target genes. Decoyscan be used as therapeutic agents, either to inhibit the expression ofgenes that are activated by a transcription factor, or to upregulategenes that are suppressed by the binding of a transcription factor.Examples of the utilization of decoy oligonucleotides can be found inMann et al., J. Clin. Invest., 2000, 106: 1071-1075, which is expresslyincorporated by reference herein, in its entirety.

miRNA Mimics

miRNA mimics represent a class of molecules that can be used to imitatethe gene modulating activity of one or more miRNAs. Thus, the term“microRNA mimic” refers to synthetic non-coding RNAs (i.e. the miRNA isnot obtained by purification from a source of the endogenous miRNA) thatare capable of entering the RNAi pathway and regulating gene expression.miRNA mimics can be designed as mature molecules (e.g. single stranded)or mimic precursors (e.g., pri- or pre-miRNAs).

In one design, miRNA mimics are double stranded molecules (e.g., with aduplex region of between about 16 and about 31 nucleotides in length)and contain one or more sequences that have identity with the maturestrand of a given miRNA. Double-stranded miRNA mimics have designssimilar to as described above for double-stranded oligonucleotides.

In one embodiment, a miRNA mimic comprises a duplex region of between 16and 31 nucleotides and one or more of the following chemicalmodification patterns: the sense strand contains 2′-O-methylmodifications of nucleotides 1 and 2 (counting from the 5′ end of thesense oligonucleotide), and all of the Cs and Us; the antisense strandmodifications can comprise 2′ F modification of all of the Cs and Us,phosphorylation of the 5′ end of the oligonucleotide, and stabilizedinternucleotide linkages associated with a 2 nucleotide 3′ overhang.

Supermirs

A supermir refers to an oligonucleotide, e.g., single stranded, doublestranded or partially double stranded, which has a nucleotide sequencethat is substantially identical to an miRNA and that is antisense withrespect to its target. This term includes oligonucleotides whichcomprise at least one non-naturally-occurring portion which functionssimilarly. In a preferred embodiment, the supermir does not include asense strand, and in another preferred embodiment, the supermir does notself-hybridize to a significant extent. An supermir featured in theinvention can have secondary structure, but it is substantiallysingle-stranded under physiological conditions. A supermir that issubstantially single-stranded is single-stranded to the extent that lessthan about 50% (e.g., less than about 40%, 30%, 20%, 10%, or 5%) of thesupermir is duplexed with itself. The supermir can include a hairpinsegment, e.g., sequence, preferably at the 3′ end can self hybridize andform a duplex region, e.g., a duplex region of at least 1, 2, 3, or 4and preferably less than 8, 7, 6, or 5 nucleotides, e.g., 5 nucleotides.The duplexed region can be connected by a linker, e.g., a nucleotidelinker, e.g., 3, 4, 5, or 6 dTs, e.g., modified dTs. In anotherembodiment the supermir is duplexed with a shorter oligo, e.g., of 5, 6,7, 8, 9, or 10 nucleotides in length, e.g., at one or both of the 3′ and5′ end or at one end and in the non-terminal or middle of the supermir.

Antimirs or miRNA Inhibitors

The terms “antimir” “microRNA inhibitor” or “miR inhibitor” aresynonymous and refer to oligonucleotides or modified oligonucleotidesthat interfere with the activity of specific miRNAs Inhibitors can adopta variety of configurations including single stranded, double stranded(RNA/RNA or RNA/DNA duplexes), and hairpin designs, in general, microRNAinhibitors comprise one or more sequences or portions of sequences thatare complementary or partially complementary with the mature strand (orstrands) of the miRNA to be targeted, in addition, the miRNA inhibitorcan also comprise additional sequences located 5′ and 3′ to the sequencethat is the reverse complement of the mature miRNA. The additionalsequences can be the reverse complements of the sequences that areadjacent to the mature miRNA in the pri-miRNA from which the maturemiRNA is derived, or the additional sequences can be arbitrary sequences(having a mixture of A, G, C, U, or dT). In some embodiments, one orboth of the additional sequences are arbitrary sequences capable offorming hairpins. Thus, in some embodiments, the sequence that is thereverse complement of the miRNA is flanked on the 5′ side and on the 3′side by hairpin structures. MicroRNA inhibitors, when double stranded,can include mismatches between nucleotides on opposite strands.Furthermore, microRNA inhibitors can be linked to conjugate moieties inorder to facilitate uptake of the inhibitor into a cell.

MicroRNA inhibitors, including hairpin miRNA inhibitors, are describedin detail in Vermeulen et al., “Double-Stranded Regions Are EssentialDesign Components Of Potent Inhibitors of RISC Function,” RNA 13:723-730 (2007) and in WO2007/095387 and WO 2008/036825 each of which isincorporated herein by reference in its entirety. A person of ordinaryskill in the art can select a sequence from the database for a desiredmiRNA and design an inhibitor useful for the methods disclosed herein.

Antagomirs

Antagomirs are RNA-like oligonucleotides that harbor variousmodifications for RNAse protection and pharmacologic properties, such asenhanced tissue and cellular uptake. They differ from normal RNA by, forexample, complete 2′-O-methylation of sugar, phosphorothioate backboneand, for example, a cholesterol-moiety at 3′-end. In a preferredembodiment, antagomir comprises a 2′-O-methylmodification at allnucleotides, a cholesterol moiety at 3′-end, two phosphorothioatebackbone linkages at the first two positions at the 5′-end and fourphosphorothioate linkages at the 3′-end of the molecule. Antagomirs canbe used to efficiently silence endogenous miRNAs by forming duplexescomprising the antagomir and endogenous miRNA, thereby preventingmiRNA-induced gene silencing. An example of antagomir-mediated miRNAsilencing is the silencing of miR-122, described in Krutzfeldt et al,Nature, 2005, 438: 685-689, which is expressly incorporated by referenceherein in its entirety.

UI Adaptors

UI adaptors inhibit polyA sites and are bifunctional oligonucleotideswith a target domain complementary to a site in the target gene'sterminal exon and a ‘UI domain’ that binds to the UI smaller nuclear RNAcomponent of the UI snRNP (Goraczniak, et al., 2008, NatureBiotechnology, 27(3), 257-263, which is expressly incorporated byreference herein, in its entirety). UI snRNP is a ribonucleoproteincomplex that functions primarily to direct early steps in spliceosomeformation by binding to the pre-mRNA exon-intron boundary (Brown andSimpson, 1998, Annu Rev Plant Physiol Plant Mol Biol 49:77-95).Nucleotides 2-11 of the 5′-end of UI snRNA base pair with the 5′-end ofsingle stranded region of the pre mRNA. In one embodiment,oligonucleotides of the invention are UI adaptors. In one embodiment,the UI adaptor can be administered in combination with at least oneother RNAi agent.

Immunostimulatory Oligonucleotides

Nucleic acids of the present invention can be immunostimulatory,including immunostimulatory oligonucleotides (single- ordouble-stranded) capable of inducing an immune response whenadministered to a subject, which can be a mammal or other patient. Theimmune response can be an innate or an adaptive immune response. Theimmune system is divided into a more innate immune system, and acquiredadaptive immune system of vertebrates, the latter of which is furtherdivided into humoral cellular components. In particular embodiments, theimmune response can be mucosal.

Immunostimulatory nucleic acids are considered to be non-sequencespecific when it is not required that they specifically bind to andreduce the expression of a target polynucleotide in order to provoke animmune response. Thus, certain immunostimulatory nucleic acids cancomprise a sequence corresponding to a region of a naturally occurringgene or mRNA, but they can still be considered non-sequence specificimmunostimulatory nucleic acids.

In one embodiment, the immunostimulatory nucleic acid or oligonucleotidecomprises at least one CpG dinucleotide. The oligonucleotide or CpGdinucleotide can be unmethylated or methylated. In another embodiment,the immunostimulatory nucleic acid comprises at least one CpGdinucleotide having a methylated cytosine. In one embodiment, thenucleic acid comprises a single CpG dinucleotide, wherein the cytosinein said CpG dinucleotide is methylated.

In another embodiment, the immunostimulatory oligonucleotide comprises aphosphate or a phosphate modification at the 5′-end. Without wishing tobe bound by theory, oligonucleotides with modified or unmodified5′-phosphates induce an anti-viral or an antibacterial response, inparticular, the induction of type I IFN, IL-18 and/or IL-1β bymodulating RIG-I.

RNA Activators

Recent studies have found that dsRNA can also activate gene expression,a mechanism that has been termed “small RNA-induced gene activation” orRNAa. See for example Li, L. C. et al. Proc Natl Acad Sci USA. (2006),103(46):17337-42 and Li L. C. (2008). “Small RNA-Mediated GeneActivation”. RNA and the Regulation of Gene Expression: A Hidden Layerof Complexity. Caister Academic Press. ISBN 978-1-904455-25-7. It hasbeen shown that dsRNAs targeting gene promoters induce potenttranscriptional activation of associated genes. Endogenous miRNA thatcause RNAa has also been found in humans. Check E. Nature (2007). 448(7156): 855-858.

Another surprising observation is that gene activation by RNAa islong-lasting. Induction of gene expression has been seen to last forover ten days. The prolonged effect of RNAa could be attributed toepigenetic changes at dsRNA target sites.

In certain embodiments, the oligonucleotide is an RNA activator, whereinoligonucleotide increases the expression of a gene. In one embodiment,increased gene expression inhibits viability, growth development, and/orreproduction.

Triplex Forming Oligonucleotides

Recent studies have shown that triplex forming oligonucleotides (TFO)can be designed which can recognize and bind topolypurine/polypyrimidine regions in double-stranded helical DNA in asequence-specific manner. These recognition rules are outline by MaherIII, L. J., et al., Science (1989) vol. 245, pp 725-730; Moser, H. E.,et al., Science (1987) vol. 238, pp 645-630; Beal, P. A., et al.,Science (1992) vol. 251, pp 1360-1363; Conney, M., et al., Science(1988) vol. 241, pp 456-459 and Hogan, M. E., et al., EP Publication375408. Modification of the oligonucleotides, such as the introductionof intercalators and backbone substitutions, and optimization of bindingconditions (pH and cation concentration) have aided in overcominginherent obstacles to TFO activity such as charge repulsion andinstability, and it was recently shown that synthetic oligonucleotidescan be targeted to specific sequences (for a recent review see Seidmanand Glazer, J Clin Invest 2003; 1 12:487-94). In general, thetriplex-forming oligonucleotide has the sequence correspondence:

oligo 3′-A G G T duplex 5′-A G C T duplex 3′-T C G A

However, it has been shown that the A-AT and G-GC triplets have thegreatest triple helical stability (Reither and Jeltsch, BMC Biochem,2002, Seρt12, Epub). The same authors have demonstrated that TFOsdesigned according to the A-AT and G-GC rule do not form non-specifictriplexes, indicating that the triplex formation is indeed sequencespecific.

Thus for any given sequence a triplex forming sequence can be devised.Triplex-forming oligonucleotides preferably are at least 15, morepreferably 25, still more preferably 30 or more nucleotides in length,up to 50 or 100 nucleotides.

Formation of the triple helical structure with the target DNA inducessteric and functional changes, blocking transcription initiation andelongation, allowing the introduction of desired sequence changes in theendogenous DNA and resulting in the specific down-regulation of geneexpression. Examples of such suppression of gene expression in cellstreated with TFOs include knockout of episomal supFG1 and endogenousHPRT genes in mammalian cells (Vasquez et al., Nucl Acids Res. 1999; 27:1176-81, and Puri, et al, J Biol Chem, 2001; 276:28991-98), and thesequence- and target specific downregulation of expression of the Ets2transcription factor, important in prostate cancer etiology (Carbone, etal, Nucl Acid Res. 2003; 31:833-43), and the pro-inflammatory ICAM-Igene (Besch et al, J Biol Chem, 2002; 277:32473-79). In addition,Vuyisich and Beal have recently shown that sequence specific TFOs canbind to dsRNA, inhibiting activity of dsRNA-dependent enzymes such asRNA-dependent kinases (Vuyisich and Beal, Nuc. Acids Res 2000;28:2369-74).

Additionally, TFOs designed according to the abovementioned principlescan induce directed mutagenesis capable of effecting DNA repair, thusproviding both down-regulation and up-regulation of expression ofendogenous genes (Seidman and Glazer, J Clin Invest 2003; 112:487-94).Detailed description of the design, synthesis and administration ofeffective TFOs can be found in U.S. Patent Application Nos. 2003 017068and 2003 0096980 to Froehler et al, and 2002 0128218 and 2002 0123476 toEmanuele et al, and U.S. Pat. No. 5,721,138 to Lawn, contents of whichare herein incorporated in their entireties.

Specific modifications are discussed in more detail below. Although, themodifications herein are described in context of an iRNA agent, thesemodifications are also amenable in modifying the carrieroligonucleotides of the invention.

The Phosphate Group

The phosphate group is a negatively charged species. The charge isdistributed equally over the two non-bridging oxygen atoms. However, thephosphate group can be modified by replacing one of the oxygens with adifferent substituent. One result of this modification to RNA phosphatebackbones can be increased resistance of the oligoribonucleotide tonucleolytic breakdown. Thus while not wishing to be bound by theory, itcan be desirable in some embodiments to introduce alterations whichresult in either an uncharged linker or a charged linker withunsymmetrical charge distribution.

Examples of modified phosphate groups include phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidates, alkyl or aryl phosphonates andphosphotriesters. Phosphorodithioates have both non-bridging oxygensreplaced by sulfur. The phosphorus center in the phosphorodithioates isachiral which precludes the formation of oligoribonucleotidesdiastereomers. Diastereomer formation can result in a preparation inwhich the individual diastereomers exhibit varying resistance tonucleases. Further, the hybridization affinity of RNA containing chiralphosphate groups can be lower relative to the corresponding unmodifiedRNA species. Thus, while not wishing to be bound by theory,modifications to both non-bridging oxygens, which eliminate the chiralcenter, e.g. phosphorodithioate formation, may be desirable in that theycannot produce diastereomer mixtures. Thus, the non-bridging oxygens canbe independently any one of S, Se, B, C, H, N, or OR (R is alkyl oraryl). Replacement of the non-bridging oxygens with sulfur is preferred.

The phosphate linker can also be modified by replacement of bridgingoxygen, (i.e. oxygen that links the phosphate to the nucleoside), withnitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates)and carbon (bridged methylenephosphonates). The replacement can occur atthe either linking oxygen or at both the linking oxygens. When thebridging oxygen is the 3′-oxygen of a nucleoside, replacement withcarbon is preferred. When the bridging oxygen is the 5′-oxygen of anucleoside, replacement with nitrogen is preferred.

Replacement of the Phosphate Group

The phosphate group can be replaced by non-phosphorus containingconnectors. While not wishing to be bound by theory, it is believed thatsince the charged phosphodiester group is the reaction center innucleolytic degradation, its replacement with neutral structural mimicsshould impart enhanced nuclease stability. Again, while not wishing tobe bound by theory, it can be desirable, in some embodiment, tointroduce alterations in which the charged phosphate group is replacedby a neutral moiety.

Examples of moieties which can replace the phosphate group includesiloxane, carbonate, carboxymethyl, carbamate, amide, thioether,ethylene oxide linker, sulfonate, sulfonamide, thioformacetal,formacetal, oxime, methyleneimino, methylenemethylimino,methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.Preferred replacements include the methylenecarbonylamino andmethylenemethylimino groups.

Modified phosphate linkages where at least one of the oxygens linked tothe phosphate has been replaced or the phosphate group has been replacedby a non-phosphorous group, are also referred to as “non phosphodiesterbackbone linkage.”

Replacement of Ribophosphate Backbone

Oligonucleotide-mimicking scaffolds can also be constructed wherein thephosphate linker and ribose sugar are replaced by nuclease resistantnucleoside or nucleotide surrogates. While not wishing to be bound bytheory, it is believed that the absence of a repetitively chargedbackbone diminishes binding to proteins that recognize polyanions (e.g.nucleases). Again, while not wishing to be bound by theory, it can bedesirable in some embodiment, to introduce alterations in which thebases are tethered by a neutral surrogate backbone. Examples include themophilino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA)nucleoside surrogates. A preferred surrogate is a PNA surrogate.

Sugar Modifications

A modified RNA can include modification of all or some of the sugargroups of the ribonucleic acid. E.g., the 2′ hydroxyl group (OH) can bemodified or replaced with a number of different “oxy” or “deoxy”substituents. While not being bound by theory, enhanced stability isexpected since the hydroxyl can no longer be deprotonated to form a2′-alkoxide ion. The 2′-alkoxide can catalyze degradation byintramolecular nucleophilic attack on the linker phosphorus atom. Again,while not wishing to be bound by theory, it can be desirable to someembodiments to introduce alterations in which alkoxide formation at the2′ position is not possible.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy oraryloxy (OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_(n)CH₂CH₂OR; “locked”nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by amethylene bridge, to the 4′ carbon of the same ribose sugar; O-AMINE(AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroaryl amino, or diheteroaryl amino, ethylene diamine,polyamino) and aminoalkoxy, O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino).It is noteworthy that oligonucleotides containing only the methoxyethylgroup (MOE), (OCH₂CH₂OCH₃, a PEG derivative), exhibit nucleasestabilities comparable to those modified with the robustphosphorothioate modification.

“Deoxy” modifications include hydrogen (i.e. deoxyribose sugars, whichare of particular relevance to the overhang portions of partially dsRNA); halo (e.g., fluoro); amino (e.g. NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroarylamino, or amino acid); NH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino), —NHC(O)R (R=alkyl, cycloalkyl,aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl;thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which maybe optionally substituted with e.g., an amino functionality. Preferredsubstitutents are 2′-methoxyethyl, 2′-OCH3, 2′-O-allyl, 2′-C-allyl, and2′-fluoro.

The sugar group can also contain one or more carbons that possess theopposite stereochemical configuration than that of the correspondingcarbon in ribose. Thus, a modified RNA can include nucleotidescontaining e.g., arabinose, as the sugar.

Modified RNAs can also include “abasic” sugars, which lack a nucleobaseat C-1′. These abasic sugars can also be further contain modificationsat one or more of the constituent sugar atoms.

To maximize nuclease resistance, the 2′ modifications can be used incombination with one or more phosphate linker modifications (e.g.,phosphorothioate). The so-called “chimeric” oligonucleotides are thosethat contain two or more different modifications.

The modification can also entail the wholesale replacement of a ribosestructure with another entity at one or more sites in the iRNA agent.

Terminal Modifications

The 3′ and 5′ ends of an oligonucleotide can be modified. Suchmodifications can be at the 3′ end, 5′ end or both ends of the molecule.They can include modification or replacement of an entire terminalphosphate or of one or more of the atoms of the phosphate group. E.g.,the 3′ and 5′ ends of an oligonucleotide can be conjugated to otherfunctional molecular entities such as labeling moieties, e.g.,fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) orprotecting groups (based e.g., on sulfur, silicon, boron or ester). Thefunctional molecular entities can be attached to the sugar through aphosphate group and/or a spacer. The terminal atom of the spacer canconnect to or replace the linking atom of the phosphate group or theC-3′ or C-5′ O, N, S or C group of the sugar. Alternatively, the spacercan connect to or replace the terminal atom of a nucleotide surrogate(e.g., PNAs). These spacers or linkers can include e.g., —(CH₂)_(n)—,—(CH₂)_(n)N—, —(CH₂)_(n)O—, —(CH₂)_(n)S—, O(CH₂CH₂O)_(n)CH₂CH₂OH (e.g.,n=3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine,thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotinand fluorescein reagents. When a spacer/phosphate-functional molecularentity-spacer/phosphate array is interposed between two strands of iRNAagents, this array can substitute for a hairpin RNA loop in ahairpin-type RNA agent. The 3′ end can be an —OH group. While notwishing to be bound by theory, it is believed that conjugation ofcertain moieties can improve transport, hybridization, and specificityproperties. Again, while not wishing to be bound by theory, it may bedesirable to introduce terminal alterations that improve nucleaseresistance. Other examples of terminal modifications include dyes,intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene,mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclicaromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificialendonucleases (e.g. EDTA), lipophilic carriers (e.g., cholesterol,cholic acid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptideconjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents,phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂,polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes,haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin,vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole,bisimidazole, histamine, imidazole clusters, acridine-imidazoleconjugates, Eu3+ complexes of tetraazamacrocycles).

Terminal modifications can be added for a number of reasons, includingas discussed elsewhere herein to modulate activity or to modulateresistance to degradation.

Terminal modifications useful for modulating activity includemodification of the 5′ end with phosphate or phosphate analogs. E.g., inpreferred embodiments iRNA agents, especially antisense strands, are 5′phosphorylated or include a phosphoryl analog at the 5′ prime terminus5′-phosphate modifications include those which are compatible with RISCmediated gene silencing. Suitable modifications include:5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylatedor non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5);5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination ofoxygen/sulfur replaced monophosphate, diphosphate and triphosphates(e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-).

Terminal modifications can also be useful for monitoring distribution,and in such cases the preferred groups to be added include fluorophores,e.g., fluorscein or an Alexa dye, e.g., Alexa 488. Terminalmodifications can also be useful for enhancing uptake, usefulmodifications for this include cholesterol. Terminal modifications canalso be useful for cross-linking an RNA agent to another moiety;modifications useful for this include mitomycin C.

Nucleobases

Adenine, guanine, cytosine and uracil are the most common bases found inRNA. These bases can be modified or replaced to provide RNA's havingimproved properties. E.g., nuclease resistant oligoribonucleotides canbe prepared with these bases or with synthetic and natural nucleobases(e.g., inosine, thymine, xanthine, hypoxanthine, nubularine,isoguanisine, or tubercidine) and any one of the above modifications.Alternatively, substituted or modified analogs of any of the abovebases, e.g., “unusual bases”, “modified bases”, “non-natural bases” and“universal bases” described herein, can be employed. Examples includewithout limitation 2-aminoadenine, 6-methyl and other alkyl derivativesof adenine and guanine, 2-propyl and other alkyl derivatives of adenineand guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine,6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyluracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other8-substituted adenines and guanines, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine, 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine,dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil,7-alkylguanine, 5-alkyl cytosine, 7-deazaadenine, N6,N6-dimethyladenine,2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole,5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil,5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil,5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil,3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine,N6-methyladenine, N6-isopentyladenine,2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylatedbases. Further purines and pyrimidines include those disclosed in U.S.Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia OfPolymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed.John Wiley & Sons, 1990, and those disclosed by Englisch et al.,Angewandte Chemie, International Edition, 1991, 30, 613.

Generally, base changes are less preferred for promoting stability, butthey can be useful for other reasons, e.g., some, e.g.,2,6-diaminopurine and 2 amino purine, are fluorescent. Modified basescan reduce target specificity. This should be taken into considerationin the design of iRNA agents.

Cationic Groups

Modifications can also include attachment of one or more cationic groupsto the sugar, base, and/or the phosphorus atom of a phosphate ormodified phosphate backbone moiety. A cationic group can be attached toany atom capable of substitution on a natural, unusual or universalbase. A preferred position is one that does not interfere withhybridization, i.e., does not interfere with the hydrogen bondinginteractions needed for base pairing. A cationic group can be attachede.g., through the C2′ position of a sugar or analogous position in acyclic or acyclic sugar surrogate. Cationic groups can include e.g.,protonated amino groups, derived from e.g., O-AMINE (AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino);aminoalkoxy, e.g., O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino, ethylene diamine, polyamino); amino (e.g. NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, diheteroaryl amino, or amino acid); orNH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroarylamino).

Exemplary Modifications and Placement within an iRNA Agent

Some modifications may preferably be included on an iRNA agent at aparticular location, e.g., at an internal position of a strand, or onthe 5′ or 3′ end of a strand of an iRNA agent. A preferred location of amodification on an iRNA agent, may confer preferred properties on theagent. For example, preferred locations of particular modifications mayconfer optimum gene silencing properties, or increased resistance toendonuclease or exonuclease activity. A modification described hereinand below may be the sole modification, or the sole type of modificationincluded on multiple ribonucleotides, or a modification can be combinedwith one or more other modifications described herein and below. Forexample, a modification on one strand of a multi-strand iRNA agent canbe different than a modification on another strand of the multi-strandiRNA agent. Similarly, two different modifications on one strand candiffer from a modification on a different strand of the iRNA agent.Other additional unique modifications, without limitation, can beincorporates into strands of the iRNA agent.

An iRNA agent may include a backbone modification to any nucleotide onan iRNA strand. For example, an iRNA agent may include aphosphorothioate linkage or P-alkyl modification in the linkages betweenone or more nucleotides of an iRNA agent. The nucleotides can beterminal nucleotides, e.g., nucleotides at the last position of a senseor antisense strand, or internal nucleotides.

An iRNA agent can include a sugar modification, e.g., a 2′ or 3′ sugarmodification. Exemplary sugar modifications include, for example, a2′-O-methylated nucleotide, a 2′-deoxy nucleotide, (e.g., a2′-deoxyfluoro nucleotide), a 2′-O-methoxyethyl nucleotide, a 2′-O-NMA,a 2′-DMAEOE, a 2′-aminopropyl, 2′-hydroxy, or a 2′-ara-fluoro or alocked nucleic acid (LNA), extended nucleic acid (ENA), hexose nucleicacid (HNA), or cyclohexene nucleic acid (CeNA). A 2′ modification ispreferably 2′-OMe, and more preferably, 2′-deoxyfluoro. When themodification is 2′-OMe, the modification is preferably on the sensestrands. When the modification is a 2′-fluoro, and the modification maybe on any strand of the iRNA agent. A 2′-ara-fluoro modification willpreferably be on the sense strands of the iRNA agent. An iRNA agent mayinclude a 3′ sugar modification, e.g., a 3′-OMe modification. Preferablya 3′-OMe modification is on the sense strand of the iRNA agent.

An iRNA agent may include a 5′-methyl-pyrimidine (e.g., a5′-methyl-uridine modification or a 5′-methyl-cytodine) modification.

The modifications described herein can be combined onto a single iRNAagent. For example, an iRNA agent may have a phosphorothioate linkageand a 2′ sugar modification, e.g., a 2′-OMe or 2′-F modification. Inanother example, an iRNA agent may include at least one 5-Me-pyrimidineand a 2′-sugar modification, e.g., a 2′-F or 2′-OMe modification.

An iRNA agent may include a nucleobase modification, such as a cationicmodification, such as a 3′-abasic cationic modification. The cationicmodification can be e.g., an alkylamino-dT (e.g., a C6 amino-dT), anallylamino conjugate, a pyrrolidine conjugate, a pthalamido, aporphyrin, or a hydroxyprolinol conjugate, on one or more of theterminal nucleotides of the iRNA agent. When an alkylamino-dT conjugateis attached to the terminal nucleotide of an iRNA agent, the conjugateis preferably attached to the 3′ end of the sense or antisense strand ofan iRNA agent. When a pyrrolidine linker is attached to the terminalnucleotide of an iRNA agent, the linker is preferably attached to the3′- or 5′-end of the sense strand, or the 3′-end of the antisensestrand. When a pyrrolidine linker is attached to the terminal nucleotideof an iRNA agent, the linker is preferably on the 3′- or 5′-end of thesense strand, and not on the 5′-end of the antisense strand.

An iRNA agent may include at least one conjugate, such as a lipophile, aterpene, a protein binding agent, a vitamin, a carbohydrate, or apeptide. For example, the conjugate can be naproxen, nitroindole (oranother conjugate that contributes to stacking interactions), folate,ibuprofen, or a C5 pyrimidine linker. The conjugate can also be aglyceride lipid conjugate (e.g., a dialkyl glyceride derivatives),vitamin E conjugate, or a thio-cholesterol. In generally, and exceptwhere noted to the contrary below, when a conjugate is on the terminalnucleotide of a sense or antisense strand, the conjugate is preferablyon the 5′ or 3′ end of the sense strand or on the 5′ end of theantisense strand, and preferably the conjugate is not on the 3′ end ofthe antisense strand.

When the conjugate is naproxen, and the conjugate is on the terminalnucleotide of a sense or antisense strand, the conjugate is preferablyon the 5′ or 3′ end of the sense or antisense strands. When theconjugate is cholesterol, and the conjugate is on the terminalnucleotide of a sense or antisense strand, the cholesterol conjugate ispreferably on the 5′ or 3′ end of the sense strand and preferably notpresent on the antisense strand. Cholesterol may be conjugated to theiRNA agent by a pyrrolidine linker, serinol linker, hydroxyprolinollinker, or disulfide linkage. A dU-cholesterol conjugate may also beconjugated to the iRNA agent by a disulfide linkage. When the conjugateis cholanic acid, and the conjugate is on the terminal nucleotide of asense or antisense strand, the cholanic acid is preferably attached tothe 5′ or 3′ end of the sense strand, or the 3′ end of the antisensestrand. In one embodiment, the cholanic acid is attached to the 3′ endof the sense strand and the 3′ end of the antisense strand.

One or more nucleotides of an iRNA agent may have a 2′-5′ linkage.Preferably, the 2′-5′ linkage is on the sense strand. When the 2′-5′linkage is on the terminal nucleotide of an iRNA agent, the 2′-5′linkage occurs on the 5′ end of the sense strand.

The iRNA agent may include an L-sugar, preferably on the sense strand,and not on the antisense strand.

The iRNA agent may include a methylphosphonate modification. When themethylphosphonate is on the terminal nucleotide of an iRNA agent, themethylphosphonate is at the 3′ end of the sense or antisense strands ofthe iRNA agent.

An iRNA agent may be modified by replacing one or more ribonucleotideswith deoxyribonucleotides. Preferably, adjacent deoxyribonucleotides arejoined by phosphorothioate linkages, and the iRNA agent does not includemore than four consecutive deoxyribonucleotides on the sense or theantisense strands.

An iRNA agent may include a difluorotoluyl (DFT) modification, e.g.,2,4-difluorotoluyl uracil, or a guanidine to inosine substitution.

The iRNA agent may include at least one 5′-uridine-adenine-3′ (5′-UA-3′)dinucleotide wherein the uridine is a 2′-modified nucleotide, or aterminal 5′-uridine-guanine-3′ (5′-UG-3′) dinucleotide, wherein the5′-uridine is a 2′-modified nucleotide, or a terminal5′-cytidine-adenine-3′ (5′-CA-3′) dinucleotide, wherein the 5′-cytidineis a 2′-modified nucleotide, or a terminal 5′-uridine-uridine-3′(5′-UU-3′) dinucleotide, wherein the 5′-uridine is a 2′-modifiednucleotide, or a terminal 5′-cytidine-cytidine-3′ (5′-CC-3′)dinucleotide, wherein the 5′-cytidine is a 2′-modified nucleotide, or aterminal 5′-cytidine-uridine-3′ (5′-CU-3′) dinucleotide, wherein the5′-cytidine is a 2′-modified nucleotide, or a terminal5′-uridine-cytidine-3′ (5′-UC-3′) dinucleotide, wherein the 5′-uridineis a 2′-modified nucleotide. The chemically modified nucleotide in theiRNA agent may be a 2′-O-methylated nucleotide. In some embodiments, themodified nucleotide can be a 2′-deoxy nucleotide, a 2′-deoxyfluoronucleotide, a 2′-O-methoxyethyl nucleotide, a 2′-O-NMA, a 2′-DMAEOE, a2′-aminopropyl, 2′-hydroxy, or a 2′-ara-fluoro, or a locked nucleic acid(LNA), extended nucleic acid (ENA), hexose nucleic acid (HNA), orcyclohexene nucleic acid (CeNA). The iRNA agents including thesemodifications are particularly stabilized against exonuclease activity,when the modified dinucleotide occurs on a terminal end of the sense orantisense strand of an iRNA agent, and are otherwise particularlystabilized against endonuclease activity.

An iRNA agent may have a single overhang, e.g., one end of the iRNAagent has a 3′ or 5′ overhang and the other end of the iRNA agent is ablunt end, or the iRNA agent may have a double overhang, e.g., both endsof the iRNA agent have a 3′ or 5′ overhang, such as a dinucleotideoverhang. In another alternative, both ends of the iRNA agent may haveblunt ends. The unpaired nucleotides may have at least onephosphorothioate dinucleotide linkage, and at least one of the unpairednucleotides may be chemically modified in the 2′-position. Thedoublestrand region of the iRNA agent may include phosphorothioatedinucleotide linkages on one or both of the sense and antisense strands.Various strands of the multi-strand iRNA agent may be connected with alinker, e.g., a chemical linker such as hexaethylene glycol linker, apoly-(oxyphosphinico-oxy-1,3-propandiol) linker, an allyl linker, or apolyethylene glycol linker.

Nuclease Resistant Compounds

An iRNA agent can include compounds which have been modified so as toinhibit degradation, e.g., by nucleases, e.g., endonucleases orexonucleases, found in the body of a subject. These compounds arereferred to herein as NRMs, or nuclease resistance promoting compoundsor modifications. In many cases these modifications will modulate otherproperties of the iRNA agent as well, e.g., the ability to interact witha protein, e.g., a transport protein, e.g., serum albumin, or a memberof the RISC (RNA-induced Silencing Complex), or the ability of the firstand second sequences to form a duplex with one another or to form aduplex with another sequence, e.g., a target molecule.

While not wishing to be bound by theory, it is believed thatmodifications of the sugar, base, and/or phosphate backbone in an iRNAagent can enhance endonuclease and exonuclease resistance, and canenhance interactions with transporter proteins and one or more of thefunctional components of the RISC complex. Preferred modifications arethose that increase exonuclease and endonuclease resistance and thusprolong the half-life of the iRNA agent prior to interaction with theRISC complex, but at the same time do not render the iRNA agentresistant to endonuclease activity in the RISC complex. Again, while notwishing to be bound by any theory, it is believed that placement of themodifications at or near the 3′ and/or 5′ end of antisense strands canresult in iRNA agents that meet the preferred nuclease resistancecriteria delineated above. Again, still while not wishing to be bound byany theory, it is believed that placement of the modifications at e.g.,the middle of a sense strand can result in iRNA agents that arerelatively less likely to undergo off-targeting.

Modifications described herein can be incorporated into any RNA andRNA-like molecule described herein, e.g., an iRNA agent, a carrieroligonucleotide. An iRNA agent may include a duplex comprising ahybridized sense and antisense strand, in which the antisense strandand/or the sense strand may include one or more of the modificationsdescribed herein. The anti sense strand may include modifications at the3′ end and/or the 5′ end and/or at one or more positions that occur 1-6(e.g., 1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand.The sense strand may include modifications at the 3′ end and/or the 5′end and/or at any one of the intervening positions between the two endsof the strand. The iRNA agent may also include a duplex comprising twohybridized antisense strands. The first and/or the second antisensestrand may include one or more of the modifications described herein.Thus, one and/or both antisense strands may include modifications at the3′ end and/or the 5′ end and/or at one or more positions that occur 1-6(e.g., 1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand.Particular configurations are discussed below.

Modifications that can be useful for producing iRNA agents that meet thepreferred nuclease resistance criteria delineated above can include oneor more of the following chemical and/or stereochemical modifications ofthe sugar, base, and/or phosphate backbone:

-   -   (i) chiral (S_(P)) thioates. Thus, preferred NRMs include        nucleotide dimers with an enriched or pure for a particular        chiral form of a modified phosphate group containing a        heteroatom at the nonbridging position, e.g., Sp or Rp, where        this is the position normally occupied by the oxygen. The        heteroatom can be S, Se, Nr₂, or Br_(a). When the heteroatom is        S, enriched or chirally pure Sp linkage is preferred. Enriched        means at least 70, 80, 90, 95, or 99% of the preferred form.        Such NRMs are discussed in more detail below;    -   (ii) attachment of one or more cationic groups to the sugar,        base, and/or the phosphorus atom of a phosphate or modified        phosphate backbone moiety. Thus, preferred NRMs include        compounds at the terminal position derivatized at a cationic        group. As the 5′ end of an antisense sequence should have a        terminal —OH or phosphate group this NRM is preferably not used        at the 5′ end of an anti-sense sequence. The group should be        attached at a position on the base which minimizes interference        with H bond formation and hybridization, e.g., away form the        face which interacts with the complementary base on the other        strand, e.g, at the 5′ position of a pyrimidine or a 7-position        of a purine. These are discussed in more detail below;    -   (iii) nonphosphate linkages at the termini. Thus, preferred NRMs        include Non-phosphate linkages, e.g., a linkage of 4 atoms which        confers greater resistance to cleavage than does a phosphate        bond. Examples include 3′ CH2-NCH₃—O—CH2-5′ and 3′        CH2-NH—(O═)—CH2-5′.;    -   (iv) 3′-bridging thiophosphates and 5′-bridging thiophosphates.        Thus, preferred NRM's can included these structures;    -   (v) L-RNA, 2′-5′ linkages, inverted linkages, a-nucleosides.        Thus, other preferred NRM's include: L nucleosides and dimeric        nucleotides derived from L-nucleosides; 2′-5′ phosphate,        non-phosphate and modified phosphate linkages (e.g.,        thiophosphates, phosphoramidates and boronophosphates); dimers        having inverted linkages, e.g., 3′-3′ or 5′-5′ linkages;        compounds having an alpha linkage at the 1′ site on the sugar,        e.g., the structures described herein having an alpha linkage;    -   (vi) conjugate groups. Thus, preferred NRM's can include e.g., a        targeting moiety or a conjugated ligand described herein        conjugated with the compound, e.g., through the sugar, base, or        backbone;    -   (vii) abasic linkages. Thus, preferred NRM's can include an        abasic compound, e.g., an abasic compound as described herein        (e.g., a nucleobaseless compound); an aromatic or heterocyclic        or polyheterocyclic aromatic compound as described herein.; and    -   (viii) 5′-phosphonates and 5′-phosphate prodrugs. Thus,        preferred NRM's include compounds, preferably at the terminal        position, e.g., the 5′ position, in which one or more atoms of        the phosphate group is derivatized with a protecting group,        which protecting group or groups, are removed as a result of the        action of a component in the subject's body, e.g, a        carboxyesterase or an enzyme present in the subject's body.        E.g., a phosphate prodrug in which a carboxy esterase cleaves        the protected molecule resulting in the production of a thioate        anion which attacks a carbon adjacent to the 0 of a phosphate        and resulting in the production of an unprotected phosphate.

One or more different NRM modifications can be introduced into an iRNAagent or into a sequence of an iRNA agent. An NRM modification can beused more than once in a sequence or in an iRNA agent. As some NRM'sinterfere with hybridization the total number incorporated, should besuch that acceptable levels of iRNA agent duplex formation aremaintained.

In some embodiments NRM modifications are introduced into the terminalthe cleavage site or in the cleavage region of a sequence (a sensestrand or sequence) which does not target a desired sequence or gene inthe subject. This can reduce off-target silencing.

REFERENCES General References

The oligoribonucleotides and oligoribonucleosides used in accordancewith this invention may be synthesized with solid phase synthesis, seefor example “Oligonucleotide synthesis, a practical approach”, Ed. M. J.Gait, IRL Press, 1984; “Oligonucleotides and Analogues, A PracticalApproach”, Ed. F. Eckstein, IRL Press, 1991 (especially Chapter 1,Modern machine-aided methods of oligodeoxyribonucleotide synthesis,Chapter 2, Oligoribonucleotide synthesis, Chapter 3,2′-O-Methyloligoribonucleotide-s: synthesis and applications, Chapter 4,Phosphorothioate oligonucleotides, Chapter 5, Synthesis ofoligonucleotide phosphorodithioates, Chapter 6, Synthesis ofoligo-2′-deoxyribonucleoside methylphosphonates, and. Chapter 7,Oligodeoxynucleotides containing modified bases. Other particularlyuseful synthetic procedures, reagents, blocking groups and reactionconditions are described in Martin, P., Helv. Chim. Acta, 1995, 78,486-504; Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48,2223-2311 and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993, 49,6123-6194, or references referred to therein.

Modification described in WO 00/44895, WO01/75164, or WO02/44321 can beused herein.

The disclosure of all publications, patents, and published patentapplications listed herein are hereby incorporated by reference.

Phosphate Group References

The preparation of phosphinate oligoribonucleotides is described in U.S.Pat. No. 5,508,270. The preparation of alkyl phosphonateoligoribonucleotides is described in U.S. Pat. No. 4,469,863. Thepreparation of phosphoramidite oligoribonucleotides is described in U.S.Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878. The preparation ofphosphotriester oligoribonucleotides is described in U.S. Pat. No.5,023,243. The preparation of borano phosphate oligoribonucleotide isdescribed in U.S. Pat. Nos. 5,130,302 and 5,177,198. The preparation of3′-Deoxy-3′-amino phosphoramidate oligoribonucleotides is described inU.S. Pat. No. 5,476,925. 3′-Deoxy-3′-methylenephosphonateoligoribonucleotides is described in An, H, et al. J. Org. Chem. 2001,66, 2789-2801. Preparation of sulfur bridged nucleotides is described inSproat et al. Nucleosides Nucleotides 1988, 7,651 and Crosstick et al.Tetrahedron Lett. 1989, 30, 4693.

Sugar Group References

Modifications to the 2′ modifications can be found in Verma, S. et al.Annu. Rev. Biochem. 1998, 67, 99-134 and all references therein.Specific modifications to the ribose can be found in the followingreferences: 2′-fluoro (Kawasaki et. al., J. Med. Chem., 1993, 36,831-841), 2′-MOE (Martin, P. Helv. Chim. Acta 1996, 79, 1930-1938),“LNA” (Wengel, J. Acc. Chem. Res. 1999, 32, 301-310).

Replacement of the Phosphate Group References

Methylenemethylimino linked oligoribonucleosides, also identified hereinas MMI linked oligoribonucleosides, methylenedimethylhydrazo linkedoligoribonucleosides, also identified herein as MDH linkedoligoribonucleosides, and methylenecarbonylamino linkedoligonucleosides, also identified herein as amide-3 linkedoligoribonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified herein as amide-4 linkedoligoribonucleosides as well as mixed backbone compounds having, as forinstance, alternating MMI and PO or PS linkages can be prepared as isdescribed in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677 and inpublished PCT applications PCT/US92/04294 and PCT/US92/04305 (publishedas WO 92/20822 WO and 92/20823, respectively). Formacetal andthioformacetal linked oligoribonucleosides can be prepared as isdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564. Ethylene oxidelinked oligoribonucleosides can be prepared as is described in U.S. Pat.No. 5,223,618. Siloxane replacements are described in Cormier, J. F. etal. Nucleic Acids Res. 1988, 16, 4583. Carbonate replacements aredescribed in Tittensor, J. R. J. Chem. Soc. C 1971, 1933. Carboxymethylreplacements are described in Edge, M. D. et al. J. Chem. Soc. PerkinTrans. 1 1972, 1991. Carbamate replacements are described in Stirchak,E. P. Nucleic Acids Res. 1989, 17, 6129.

Replacement of the Phosphate-Ribose Backbone References

Cyclobutyl sugar surrogate compounds can be prepared as is described inU.S. Pat. No. 5,359,044. Pyrrolidine sugar surrogate can be prepared asis described in U.S. Pat. No. 5,519,134. Morpholino sugar surrogates canbe prepared as is described in U.S. Pat. Nos. 5,142,047 and 5,235,033,and other related patent disclosures. Peptide Nucleic Acids (PNAs) areknown per se and can be prepared in accordance with any of the variousprocedures referred to in Peptide Nucleic Acids (PNA): Synthesis,Properties and Potential Applications, Bioorganic & Medicinal Chemistry,1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat.No. 5,539,083.

Terminal Modification References

Terminal modifications are described in Manoharan, M. et al. Antisenseand Nucleic Acid Drug Development 12, 103-128 (2002) and referencestherein.

Bases References

N-2 substituted purine nucleoside amidites can be prepared as isdescribed in U.S. Pat. No. 5,459,255. 3-Deaza purine nucleoside amiditescan be prepared as is described in U.S. Pat. No. 5,457,191.5,6-Substituted pyrimidine nucleoside amidites can be prepared as isdescribed in U.S. Pat. No. 5,614,617. 5-Propynyl pyrimidine nucleosideamidites can be prepared as is described in U.S. Pat. No. 5,484,908.Additional references are disclosed in the above section on basemodifications.

Oligonucleotide Production

The oligonucleotide compounds of the invention can be prepared usingsolution-phase or solid-phase organic synthesis. Organic synthesisoffers the advantage that the oligonucleotide strands comprisingnon-natural or modified nucleotides can be easily prepared. Any othermeans for such synthesis known in the art may additionally oralternatively be employed. It is also known to use similar techniques toprepare other oligonucleotides, such as the phosphorothioates,phosphorodithioates and alkylated derivatives. The double-strandedoligonucleotide compounds of the invention may be prepared using atwo-step procedure. First, the individual strands of the double-strandedmolecule are prepared separately. Then, the component strands areannealed.

Regardless of the method of synthesis, the oligonucleotide can beprepared in a solution (e.g., an aqueous and/or organic solution) thatis appropriate for formulation. For example, the iRNA preparation can beprecipitated and redissolved in pure double-distilled water, andlyophilized. The dried iRNA can then be resuspended in a solutionappropriate for the intended formulation process.

Teachings regarding the synthesis of particular modifiedoligonucleotides may be found in the following U.S. patents or pendingpatent applications: U.S. Pat. Nos. 5,138,045 and 5,218,105, drawn topolyamine conjugated oligonucleotides; U.S. Pat. No. 5,212,295, drawn tocompounds for the preparation of oligonucleotides having chiralphosphorus linkages; U.S. Pat. Nos. 5,378,825 and 5,541,307, drawn tooligonucleotides having modified backbones; U.S. Pat. No. 5,386,023,drawn to backbone-modified oligonucleotides and the preparation thereofthrough reductive coupling; U.S. Pat. No. 5,457,191, drawn to modifiednucleobases based on the 3-deazapurine ring system and methods ofsynthesis thereof; U.S. Pat. No. 5,459,255, drawn to modifiednucleobases based on N-2 substituted purines; U.S. Pat. No. 5,521,302,drawn to processes for preparing oligonucleotides having chiralphosphorus linkages; U.S. Pat. No. 5,539,082, drawn to peptide nucleicacids; U.S. Pat. No. 5,554,746, drawn to oligonucleotides having.beta.-lactam backbones; U.S. Pat. No. 5,571,902, drawn to methods andmaterials for the synthesis of oligonucleotides; U.S. Pat. No.5,578,718, drawn to nucleosides having alkylthio groups, wherein suchgroups may be used as linkers to other moieties attached at any of avariety of positions of the nucleoside; U.S. Pat. Nos. 5,587,361 and5,599,797, drawn to oligonucleotides having phosphorothioate linkages ofhigh chiral purity; U.S. Pat. No. 5,506,351, drawn to processes for thepreparation of 2′-O-alkyl guanosine and related compounds, including2,6-diaminopurine compounds; U.S. Pat. No. 5,587,469, drawn tooligonucleotides having N-2 substituted purines; U.S. Pat. No.5,587,470, drawn to oligonucleotides having 3-deazapurines; U.S. Pat.No. 5,223,168, and U.S. Pat. No. 5,608,046, both drawn to conjugated4′-desmethyl nucleoside analogs; U.S. Pat. Nos. 5,602,240, and5,610,289, drawn to backbone-modified oligonucleotide analogs; and U.S.Pat. Nos. 6,262,241, and 5,459,255, drawn to, inter alia, methods ofsynthesizing 2′-fluoro-oligonucleotides.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one type of modification maybe incorporated in a single oligonucleotide compound or even in a singlenucleotide thereof.

Routes of Delivery

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions and methods can be practiced with other oligonucleotide ofthe invention, e.g., modified iRNA agents, antisense, antagomirs,aptamers, ribozymes, and such practice is within the invention. Acomposition that includes an iRNA can be delivered to a subject by avariety of routes. Exemplary routes include: intravenous, topical,rectal, anal, vaginal, nasal, pulmonary, ocular.

The iRNA molecules of the invention can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically include one or more species of iRNA and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic, vaginal, rectal,intranasal, transdermal), oral or parenteral. Parenteral administrationincludes intravenous drip, subcutaneous, intraperitoneal orintramuscular injection, or intrathecal or intraventricularadministration.

The route and site of administration may be chosen to enhance targeting.For example, to target muscle cells, intramuscular injection into themuscles of interest would be a logical choice. Lung cells might betargeted by administering the iRNA in aerosol form. The vascularendothelial cells could be targeted by coating a balloon catheter withthe iRNA and mechanically introducing the DNA.

Formulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.Coated condoms, gloves and the like may also be useful.

Compositions for oral administration include powders or granules,suspensions or solutions in water, syrups, elixirs or non-aqueous media,tablets, capsules, lozenges, or troches. In the case of tablets,carriers that can be used include lactose, sodium citrate and salts ofphosphoric acid. Various disintegrants such as starch, and lubricatingagents such as magnesium stearate, sodium lauryl sulfate and talc, arecommonly used in tablets. For oral administration in capsule form,useful diluents are lactose and high molecular weight polyethyleneglycols. When aqueous suspensions are required for oral use, the nucleicacid compositions can be combined with emulsifying and suspendingagents. If desired, certain sweetening and/or flavoring agents can beadded.

Compositions for intrathecal or intraventricular administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives.

Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives. Intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir. Forintravenous use, the total concentration of solutes should be controlledto render the preparation isotonic.

For ocular administration, ointments or droppable liquids may bedelivered by ocular delivery systems known to the art such asapplicators or eye droppers. Such compositions can include mucomimeticssuch as hyaluronic acid, chondroitin sulfate, hydroxypropylmethylcellulose or poly(vinyl alcohol), preservatives such as sorbicacid, EDTA or benzylchronium chloride, and the usual quantities ofdiluents and/or carriers.

Topical Delivery

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions and methods can be practiced with other oligonucleotides ofthe invention, e.g., modified iRNA agents, antisense, aptamer, antagomirand ribozyme, and such practice is within the invention. In a preferredembodiment, an iRNA agent is delivered to a subject via topicaladministration. “Topical administration” refers to the delivery to asubject by contacting the formulation directly to a surface of thesubject. The most common form of topical delivery is to the skin, but acomposition disclosed herein can also be directly applied to othersurfaces of the body, e.g., to the eye, a mucous membrane, to surfacesof a body cavity or to an internal surface. As mentioned above, the mostcommon topical delivery is to the skin. The term encompasses severalroutes of administration including, but not limited to, topical andtransdermal. These modes of administration typically include penetrationof the skin's permeability barrier and efficient delivery to the targettissue or stratum. Topical administration can be used as a means topenetrate the epidermis and dermis and ultimately achieve systemicdelivery of the composition. Topical administration can also be used asa means to selectively deliver oligonucleotides to the epidermis ordermis of a subject, or to specific strata thereof, or to an underlyingtissue.

The term “skin,” as used herein, refers to the epidermis and/or dermisof an animal. Mammalian skin consists of two major, distinct layers. Theouter layer of the skin is called the epidermis. The epidermis iscomprised of the stratum corneum, the stratum granulosum, the stratumspinosum, and the stratum basale, with the stratum corneum being at thesurface of the skin and the stratum basale being the deepest portion ofthe epidermis. The epidermis is between 50 μm and 0.2 mm thick,depending on its location on the body.

Beneath the epidermis is the dermis, which is significantly thicker thanthe epidermis. The dermis is primarily composed of collagen in the formof fibrous bundles. The collagenous bundles provide support for, interalia, blood vessels, lymph capillaries, glands, nerve endings andimmunologically active cells.

One of the major functions of the skin as an organ is to regulate theentry of substances into the body. The principal permeability barrier ofthe skin is provided by the stratum corneum, which is formed from manylayers of cells in various states of differentiation. The spaces betweencells in the stratum corneum is filled with different lipids arranged inlattice-like formations that provide seals to further enhance the skinspermeability barrier.

The permeability barrier provided by the skin is such that it is largelyimpermeable to molecules having molecular weight greater than about 750Da. For larger molecules to cross the skin's permeability barrier,mechanisms other than normal osmosis must be used.

Several factors determine the permeability of the skin to administeredagents. These factors include the characteristics of the treated skin,the characteristics of the delivery agent, interactions between both thedrug and delivery agent and the drug and skin, the dosage of the drugapplied, the form of treatment, and the post treatment regimen. Toselectively target the epidermis and dermis, it is sometimes possible toformulate a composition that comprises one or more penetration enhancersthat will enable penetration of the drug to a preselected stratum.

Transdermal delivery is a valuable route for the administration of lipidsoluble therapeutics. The dermis is more permeable than the epidermisand therefore absorption is much more rapid through abraded, burned ordenuded skin. Inflammation and other physiologic conditions thatincrease blood flow to the skin also enhance transdermal adsorption.Absorption via this route may be enhanced by the use of an oily vehicle(inunction) or through the use of one or more penetration enhancers.Other effective ways to deliver a composition disclosed herein via thetransdermal route include hydration of the skin and the use ofcontrolled release topical patches. The transdermal route provides apotentially effective means to deliver a composition disclosed hereinfor systemic and/or local therapy.

In addition, iontophoresis (transfer of ionic solutes through biologicalmembranes under the influence of an electric field) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 163),phonophoresis or sonophoresis (use of ultrasound to enhance theabsorption of various therapeutic agents across biological membranes,notably the skin and the cornea) (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 166), and optimization ofvehicle characteristics relative to dose position and retention at thesite of administration (Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 168) may be useful methods for enhancing thetransport of topically applied compositions across skin and mucosalsites.

The compositions and methods provided may also be used to examine thefunction of various proteins and genes in vitro in cultured or preserveddermal tissues and in animals. The invention can be thus applied toexamine the function of any gene. The methods of the invention can alsobe used therapeutically or prophylactically. For example, for thetreatment of animals that are known or suspected to suffer from diseasessuch as psoriasis, lichen planus, toxic epidermal necrolysis, ertythemamultiforme, basal cell carcinoma, squamous cell carcinoma, malignantmelanoma, Paget's disease, Kaposi's sarcoma, pulmonary fibrosis, Lymedisease and viral, fungal and bacterial infections of the skin.

Pulmonary Delivery

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions and methods can be practiced with other oligonucleotides ofthe invention, e.g., modified iRNA agents, antisense, aptamer, antagomirand ribozyme, and such practice is within the invention. A compositionthat includes an iRNA agent, e.g., a double-stranded iRNA agent, can beadministered to a subject by pulmonary delivery. Pulmonary deliverycompositions can be delivered by inhalation by the patient of adispersion so that the composition, preferably iRNA, within thedispersion can reach the lung where it can be readily absorbed throughthe alveolar region directly into blood circulation. Pulmonary deliverycan be effective both for systemic delivery and for localized deliveryto treat diseases of the lungs.

Pulmonary delivery can be achieved by different approaches, includingthe use of nebulized, aerosolized, micellular and dry powder-basedformulations. Delivery can be achieved with liquid nebulizers,aerosol-based inhalers, and dry powder dispersion devices. Metered-dosedevices are preferred. One of the benefits of using an atomizer orinhaler is that the potential for contamination is minimized because thedevices are self contained. Dry powder dispersion devices, for example,deliver drugs that may be readily formulated as dry powders. An iRNAcomposition may be stably stored as lyophilized or spray-dried powdersby itself or in combination with suitable powder carriers. The deliveryof a composition for inhalation can be mediated by a dosing timingelement which can include a timer, a dose counter, time measuringdevice, or a time indicator which when incorporated into the deviceenables dose tracking, compliance monitoring, and/or dose triggering toa patient during administration of the aerosol medicament.

The term “powder” means a composition that consists of finely dispersedsolid particles that are free flowing and capable of being readilydispersed in an inhalation device and subsequently inhaled by a subjectso that the particles reach the lungs to permit penetration into thealveoli. Thus, the powder is said to be “respirable.” Preferably theaverage particle size is less than about 10 μm in diameter preferablywith a relatively uniform spheroidal shape distribution. More preferablythe diameter is less than about 7.5 μm and most preferably less thanabout 5.0 μm. Usually the particle size distribution is between about0.1 μm and about 5 μm in diameter, particularly about 0.3 μm to about 5μm.

The term “dry” means that the composition has a moisture content belowabout 10% by weight (% w) water, usually below about 5% w and preferablyless it than about 3% w. A dry composition can be such that theparticles are readily dispersible in an inhalation device to form anaerosol.

The term “therapeutically effective amount” is the amount present in thecomposition that is needed to provide the desired level of drug in thesubject to be treated to give the anticipated physiological response.

The term “physiologically effective amount” is that amount delivered toa subject to give the desired palliative or curative effect.

The term “pharmaceutically acceptable carrier” means that the carriercan be taken into the lungs with no significant adverse toxicologicaleffects on the lungs.

The types of pharmaceutical excipients that are useful as carrierinclude stabilizers such as human serum albumin (HSA), bulking agentssuch as carbohydrates, amino acids and polypeptides; pH adjusters orbuffers; salts such as sodium chloride; and the like. These carriers maybe in a crystalline or amorphous form or may be a mixture of the two.

Bulking agents that are particularly valuable include compatiblecarbohydrates, polypeptides, amino acids or combinations thereof.Suitable carbohydrates include monosaccharides such as galactose,D-mannose, sorbose, and the like; disaccharides, such as lactose,trehalose, and the like; cyclodextrins, such as2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such asraffinose, maltodextrins, dextrans, and the like; alditols, such asmannitol, xylitol, and the like. A preferred group of carbohydratesincludes lactose, threhalose, raffinose maltodextrins, and mannitol.Suitable polypeptides include aspartame. Amino acids include alanine andglycine, with glycine being preferred.

Additives, which are minor components of the composition of thisinvention, may be included for conformational stability during spraydrying and for improving dispersibility of the powder. These additivesinclude hydrophobic amino acids such as tryptophan, tyrosine, leucine,phenylalanine, and the like.

Suitable pH adjusters or buffers include organic salts prepared fromorganic acids and bases, such as sodium citrate, sodium ascorbate, andthe like; sodium citrate is preferred.

Pulmonary administration of a micellar iRNA formulation may be achievedthrough metered dose spray devices with propellants such astetrafluoroethane, heptafluoroethane, dimethylfluoropropane,tetrafluoropropane, butane, isobutane, dimethyl ether and other non-CFCand CFC propellants.

Oral or Nasal Delivery

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions and methods can be practiced with other oligonucleotides ofthe invention, e.g., modified iRNA agents, antisense, aptamer, antagomirand ribozyme, and such practice is within the invention. Both the oraland nasal membranes offer advantages over other routes ofadministration. For example, drugs administered through these membraneshave a rapid onset of action, provide therapeutic plasma levels, avoidfirst pass effect of hepatic metabolism, and avoid exposure of the drugto the hostile gastrointestinal (GI) environment. Additional advantagesinclude easy access to the membrane sites so that the drug can beapplied, localized and removed easily.

In oral delivery, compositions can be targeted to a surface of the oralcavity, e.g., to sublingual mucosa which includes the membrane ofventral surface of the tongue and the floor of the mouth or the buccalmucosa which constitutes the lining of the cheek. The sublingual mucosais relatively permeable thus giving rapid absorption and acceptablebioavailability of many drugs. Further, the sublingual mucosa isconvenient, acceptable and easily accessible.

The ability of molecules to permeate through the oral mucosa appears tobe related to molecular size, lipid solubility and peptide proteinionization. Small molecules, less than 1000 daltons appear to crossmucosa rapidly. As molecular size increases, the permeability decreasesrapidly. Lipid soluble compounds are more permeable than non-lipidsoluble molecules. Maximum absorption occurs when molecules areun-ionized or neutral in electrical charges. Therefore charged moleculespresent the biggest challenges to absorption through the oral mucosae.

A pharmaceutical composition of iRNA may also be administered to thebuccal cavity of a human being by spraying into the cavity, withoutinhalation, from a metered dose spray dispenser, a mixed micellarpharmaceutical formulation as described above and a propellant. In oneembodiment, the dispenser is first shaken prior to spraying thepharmaceutical formulation and propellant into the buccal cavity.

Devices

For ease of exposition the devices, formulations, compositions andmethods in this section are discussed largely with regard to unmodifiediRNA agents. It should be understood, however, that these formulations,compositions and methods can be practiced with other oligonucleotides ofthe invention, e.g., modified iRNA agents, antisense, aptamer, antagomirand ribozyme, and such practice is within the invention. An iRNA agent,e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor,e.g., a larger iRNA agent which can be processed into a sRNA agent, or aDNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, orsRNA agent, or precursor thereof) can be disposed on or in a device,e.g., a device which implanted or otherwise placed in a subject.Exemplary devices include devices which are introduced into thevasculature, e.g., devices inserted into the lumen of a vascular tissue,or which devices themselves form a part of the vasculature, includingstents, catheters, heart valves, and other vascular devices. Thesedevices, e.g., catheters or stents, can be placed in the vasculature ofthe lung, heart, or leg.

Other devices include non-vascular devices, e.g., devices implanted inthe peritoneum, or in organ or glandular tissue, e.g., artificialorgans. The device can release a therapeutic substance in addition to aiRNA, e.g., a device can release insulin.

Other devices include artificial joints, e.g., hip joints, and otherorthopedic implants.

In one embodiment, unit doses or measured doses of a composition thatincludes iRNA are dispensed by an implanted device. The device caninclude a sensor that monitors a parameter within a subject. Forexample, the device can include pump, e.g., and, optionally, associatedelectronics.

Tissue, e.g., cells or organs, such as the kidney, can be treated withan iRNA agent ex vivo and then administered or implanted in a subject.

The tissue can be autologous, allogeneic, or xenogeneic tissue. Forexample, tissue (e.g., kidney) can be treated to reduce graft v. hostdisease. In other embodiments, the tissue is allogeneic and the tissueis treated to treat a disorder characterized by unwanted gene expressionin that tissue, such as in the kidney. In another example, tissuecontaining hematopoietic cells, e.g., bone marrow hematopoietic cells,can be treated to inhibit unwanted cell proliferation.

Introduction of treated tissue, whether autologous or transplant, can becombined with other therapies.

In some implementations, the iRNA treated cells are insulated from othercells, e.g., by a semi-permeable porous barrier that prevents the cellsfrom leaving the implant, but enables molecules from the body to reachthe cells and molecules produced by the cells to enter the body. In oneembodiment, the porous barrier is formed from alginate.

In one embodiment, a contraceptive device is coated with or contains aniRNA agent. Exemplary devices include condoms, diaphragms, IUD(implantable uterine devices, sponges, vaginal sheaths, and birthcontrol devices. In one embodiment, the iRNA is chosen to inactive spermor egg. In another embodiment, the iRNA is chosen to be complementary toa viral or pathogen RNA, e.g., an RNA of an STD. In some instances, theiRNA composition can include a spermicidal agent.

Formulations

The iRNA agents described herein can be formulated for administration toa subject. For ease of exposition the formulations, compositions andmethods in this section are discussed largely with regard to unmodifiediRNA agents. It should be understood, however, that these formulations,compositions and methods can be practiced with other oligonucleotides ofthe invention, e.g., modified iRNA agents, antisense, aptamer, antagomirand ribozyme, and such practice is within the invention.

A formulated iRNA composition can assume a variety of states. In someexamples, the composition is at least partially crystalline, uniformlycrystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10%water). In another example, the iRNA is in an aqueous phase, e.g., in asolution that includes water.

The aqueous phase or the crystalline compositions can, e.g., beincorporated into a delivery vehicle, e.g., a liposome (particularly forthe aqueous phase) or a particle (e.g., a microparticle as can beappropriate for a crystalline composition). Generally, the iRNAcomposition is formulated in a manner that is compatible with theintended method of administration.

In particular embodiments, the composition is prepared by at least oneof the following methods: spray drying, lyophilization, vacuum drying,evaporation, fluid bed drying, or a combination of these techniques; orsonication with a lipid, freeze-drying, condensation and otherself-assembly.

An iRNA preparation can be formulated in combination with another agent,e.g., another therapeutic agent or an agent that stabilizes a iRNA,e.g., a protein that complexes with iRNA to form an iRNP. Still otheragents include chelators, e.g., EDTA (e.g., to remove divalent cationssuch as Mg²⁺), salts, RNAse inhibitors (e.g., a broad specificity RNAseinhibitor such as RNAsin) and so forth.

In one embodiment, the iRNA preparation includes another iRNA agent,e.g., a second iRNA that can mediated RNAi with respect to a secondgene, or with respect to the same gene. Still other preparation caninclude at least 3, 5, ten, twenty, fifty, or a hundred or moredifferent iRNA species. Such iRNAs can mediated RNAi with respect to asimilar number of different genes.

In one embodiment, the iRNA preparation includes at least a secondtherapeutic agent (e.g., an agent other than a RNA or a DNA). Forexample, an iRNA composition for the treatment of a viral disease, e.g.HIV, might include a known antiviral agent (e.g., a protease inhibitoror reverse transcriptase inhibitor). In another example, an iRNAcomposition for the treatment of a cancer might further comprise achemotherapeutic agent.

Other formulations amenable to the present invention are described inU.S. provisional application Ser. Nos. 61/018,616, filed Jan. 2, 2008;61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008;61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCTapplication no PCT/US2007/080331, filed Oct. 3, 2007 also describesformulations that are amenable to the present invention.

Pharmaceutical Compositions

In one embodiment, the invention relates to a pharmaceutical compositioncontaining an oligonucleotide of the invention e.g. an iRNA agent, asdescribed in the preceding sections, and a pharmaceutically acceptablecarrier, as described below. A pharmaceutical composition including themodified iRNA agent is useful for treating a disease caused byexpression of a target gene. In this aspect of the invention, the iRNAagent of the invention is formulated as described below. Thepharmaceutical composition is administered in a dosage sufficient toinhibit expression of the target gene.

The pharmaceutical compositions of the present invention areadministered in dosages sufficient to inhibit the expression or activityof the target gene. Compositions containing the iRNA agent of theinvention can be administered at surprisingly low dosages. A maximumdosage of 5 mg iRNA agent per kilogram body weight per day may besufficient to inhibit or completely suppress the expression or activityof the target gene.

In general, a suitable dose of modified iRNA agent will be in the rangeof 0.001 to 500 milligrams per kilogram body weight of the recipient perday (e.g., about 1 microgram per kilogram to about 500 milligrams perkilogram, about 100 micrograms per kilogram to about 100 milligrams perkilogram, about 1 milligrams per kilogram to about 75 milligrams perkilogram, about 10 micrograms per kilogram to about 50 milligrams perkilogram, or about 1 microgram per kilogram to about 50 micrograms perkilogram). The pharmaceutical composition may be administered once perday, or the iRNA agent may be administered as two, three, four, five,six or more sub-doses at appropriate intervals throughout the day. Inthat case, the iRNA agent contained in each sub-dose must becorrespondingly smaller in order to achieve the total daily dosage. Thedosage unit can also be compounded for delivery over several days, e.g.,using a conventional sustained release formulation which providessustained release of the iRNA agent over a several day period. Sustainedrelease formulations are well known in the art. In this embodiment, thedosage unit contains a corresponding multiple of the daily dose.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the infection or disease, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual iRNA agent encompassed by theinvention can be made using conventional methodologies or on the basisof in vivo testing using an appropriate animal model, as describedelsewhere herein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases. For example, mouse repositories canbe found at The Jackson Laboratory, Charles River Laboratories, Taconic,Harlan, Mutant Mouse Regional Resource Centers (MMRRC) National Networkand at the European Mouse Mutant Archive. Such models may be used for invivo testing of iRNA agent, as well as for determining a therapeuticallyeffective dose.

The pharmaceutical compositions encompassed by the invention may beadministered by any means known in the art including, but not limited tooral or parenteral routes, including intravenous, intramuscular,intraperitoneal, subcutaneous, transdermal, airway (aerosol), ocular,rectal, vaginal and topical (including buccal and sublingual)administration. In preferred embodiments, the pharmaceuticalcompositions are administered by intravenous or intraparenteral infusionor injection. The pharmaceutical compositions can also be administeredintraparenchymally, intrathecally, and/or by stereotactic injection.

For oral administration, the iRNA agent useful in the invention willgenerally be provided in the form of tablets or capsules, as a powder orgranules, or as an aqueous solution or suspension.

Tablets for oral use may include the active ingredients mixed withpharmaceutically acceptable excipients such as inert diluents,disintegrating agents, binding agents, lubricating agents, sweeteningagents, flavoring agents, coloring agents and preservatives. Suitableinert diluents include sodium and calcium carbonate, sodium and calciumphosphate, and lactose, while corn starch and alginic acid are suitabledisintegrating agents. Binding agents may include starch and gelatin,while the lubricating agent, if present, will generally be magnesiumstearate, stearic acid or talc. If desired, the tablets may be coatedwith a material such as glyceryl monostearate or glyceryl distearate, todelay absorption in the gastrointestinal tract.

Capsules for oral use include hard gelatin capsules in which the activeingredient is mixed with a solid diluent, and soft gelatin capsuleswherein the active ingredient is mixed with water or an oil such aspeanut oil, liquid paraffin or olive oil.

For intramuscular, intraperitoneal, subcutaneous and intravenous use,the pharmaceutical compositions of the invention will generally beprovided in sterile aqueous solutions or suspensions, buffered to anappropriate pH and isotonicity. Suitable aqueous vehicles includeRinger's solution and isotonic sodium chloride. In a preferredembodiment, the carrier consists exclusively of an aqueous buffer. Inthis context, “exclusively” means no auxiliary agents or encapsulatingsubstances are present which might affect or mediate uptake of iRNAagent in the cells that harbor the target gene or virus. Such substancesinclude, for example, micellar structures, such as liposomes or capsids,as described below. Although microinjection, lipofection, viruses,viroids, capsids, capsoids, or other auxiliary agents are required tointroduce iRNA agent into cell cultures, surprisingly these methods andagents are not necessary for uptake of iRNA agent in vivo. The iRNAagent of the present invention are particularly advantageous in thatthey do not require the use of an auxiliary agent to mediate uptake ofthe iRNA agent into the cell, many of which agents are toxic orassociated with deleterious side effects. Aqueous suspensions accordingto the invention may include suspending agents such as cellulosederivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth,and a wetting agent such as lecithin. Suitable preservatives for aqueoussuspensions include ethyl and n-propyl p-hydroxybenzoate.

The pharmaceutical compositions can also include encapsulatedformulations to protect the iRNA agent against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811; PCT publication WO 91/06309; and European patent publicationEP-A-43075, which are incorporated by reference herein.

Toxicity and therapeutic efficacy of iRNA agent can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.iRNA agents that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosages ofcompositions of the invention are preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anyiRNA agent used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range of the iRNA agent or, when appropriate, of thepolypeptide product of a target sequence (e.g., achieving a decreasedconcentration of the polypeptide) that includes the IC50 (i.e., theconcentration of the test iRNA agent which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

In addition to their administration individually or as a plurality, asdiscussed above, iRNA agents relating to the invention can beadministered in combination with other known agents effective intreating viral infections and diseases. In any event, the administeringphysician can adjust the amount and timing of iRNA agent administrationon the basis of results observed using standard measures of efficacyknown in the art or described herein.

Combination Therapy

In one aspect, composition of the invention can be used in combinationtherapy. The term “combination therapy” includes the administration ofthe subject compounds in further combination with other biologicallyactive ingredients (such as, but not limited to, a second and differentantineoplastic agent) and non-drug therapies (such as, but not limitedto, surgery or radiation treatment). For instance, the compounds of theinvention can be used in combination with other pharmaceutically activecompounds, preferably compounds that are able to enhance the effect ofthe compounds of the invention. The compounds of the invention can beadministered simultaneously (as a single preparation or separatepreparation) or sequentially to the other drug therapy. In general, acombination therapy envisions administration of two or more drugs duringa single cycle or course of therapy.

In one aspect of the invention, the subject compounds may beadministered in combination with one or more separate agents thatmodulate protein kinases involved in various disease states. Examples ofsuch kinases may include, but are not limited to: serine/threoninespecific kinases, receptor tyrosine specific kinases and non-receptortyrosine specific kinases. Serine/threonine kinases include mitogenactivated protein kinases (MAPK), meiosis specific kinase (MEK), RAF andaurora kinase. Examples of receptor kinase families include epidermalgrowth factor receptor (EGFR) (e.g. HER2/neu, HER3, HERO, ErbB, ErbB2,ErbB3, ErbB4, Xmrk, DER, Let23); fibroblast growth factor (FGF) receptor(e.g. FGF-R1, GFF-R2/BEK/CEK3, FGF-R3/CEK2, FGF-R4/TKF, KGF-R);hepatocyte growth/scatter factor receptor (HGFR) (e.g, MET, RON, SEA,SEX); insulin receptor (e.g. IGFI-R); Eph (e.g. CEK5, CEK8, EBK, ECK,EEK, EHK-I, EHK-2, ELK, EPH, ERK, HEK, MDK2, MDK5, SEK); AxI (e.g.Mer/Nyk, Rse); RET; and platelet-derived growth factor receptor (PDGFR)(e.g. PDGFa-R, PDGβ-R, CSF1-R/FMS, SCF-R/C-KIT, VEGF-R/FLT, NEK/FLK1,FLT3/FLK2/STK-1). Non-receptor tyrosine kinase families include, but arenot limited to, BCR-ABL (e.g. p43^(ab1), ARG); BTK (e.g. ITK/EMT, TEC);CSK, FAK, FPS, JAK, SRC, BMX, FER, CDK and SYK.

In another aspect of the invention, the subject compounds may beadministered in combination with one or more agents that modulatenon-kinase biological targets or processes. Such targets include histonedeacetylases (HDAC), DNA methyltransferase (DNMT), heat shock proteins(e.g. HSP90), and proteosomes.

In one embodiment, subject compounds may be combined with antineoplasticagents (e.g. small molecules, monoclonal antibodies, antisense RNA, andfusion proteins) that inhibit one or more biological targets such asZolinza, Tarceva, Iressa, Tykerb, Gleevec, Sutent, Sprycel, Nexavar,Sorafinib, CNF2024, RG108, BMS387032, Affmitak, Avastin, Herceptin,Erbitux, AG24322, PD325901, ZD6474, PD 184322,

Obatodax, ABT737 and AEE788. Such combinations may enhance therapeuticefficacy over efficacy achieved by any of the agents alone and mayprevent or delay the appearance of resistant mutational variants.

In certain preferred embodiments, the compounds of the invention areadministered in combination with a chemotherapeutic agent.Chemotherapeutic agents encompass a wide range of therapeutic treatmentsin the field of oncology. These agents are administered at variousstages of the disease for the purposes of shrinking tumors, destroyingremaining cancer cells left over after surgery, inducing remission,maintaining remission and/or alleviating symptoms relating to the canceror its treatment. Examples of such agents include, but are not limitedto, alkylating agents such as mustard gas derivatives

(Mechlorethamine, cylophosphamide, chlorambucil, melphalan, ifosfamide),ethylenimines (thiotepa, hexamethylmelanine), Alkylsulfonates(Busulfan), Hydrazines and Triazines (Altretamine, Procarbazine,Dacarbazine and Temozolomide), Nitrosoureas (Carmustine, Lomustine andStreptozocin), Ifosfamide and metal salts (Carboplatin, Cisplatin, andOxaliplatin); plant alkaloids such as Podophyllotoxins (Etoposide andTenisopide), Taxanes (Paclitaxel and Docetaxel), Vinca alkaloids(Vincristine, Vinblastine, Vindesine and Vinorelbine), and Camptothecananalogs (Irinotecan and Topotecan); anti-tumor antibiotics such asChromomycins (Dactinomycin and Plicamycin), Anthracyclines (Doxorubicin,Daunorubicin, Epirubicin, Mitoxantrone, Valrubicin and Idarubicin), andmiscellaneous antibiotics such as Mitomycin, Actinomycin and Bleomycin;anti-metabolites such as folic acid antagonists (Methotrexate,Pemetrexed, Raltitrexed, Aminopterin), pyrimidine antagonists(5-Fluorouracil, Floxuridine, Cytarabine, Capecitabine, andGemcitabine), purine antagonists (6-Mercaptopurine and 6-Thioguanine)and adenosine deaminase inhibitors (Cladribine, Fludarabine,Mercaptopurine, Clofarabine, Thioguanine, Nelarabine and Pentostatin);topoisomerase inhibitors such as topoisomerase I inhibitors (Ironotecan,topotecan) and topoisomerase II inhibitors (Amsacrine, etoposide,etoposide phosphate, teniposide); monoclonal antibodies (Alemtuzumab,Gemtuzumab ozogamicin, Rituximab, Trastuzumab, Ibritumomab Tioxetan,Cetuximab, Panitumumab, Tositumomab,

Bevacizumab); and miscellaneous anti-neoplasties such as ribonucleotidereductase inhibitors (Hydroxyurea); adrenocortical steroid inhibitor(Mitotane); enzymes (Asparaginase and Pegaspargase); anti-microtubuleagents (Estramustine); and retinoids (Bexarotene, Isotretinoin,Tretinoin (ATRA). In certain preferred embodiments, the compounds of theinvention are administered in combination with a chemoprotective agent.Chemoprotective agents act to protect the body or minimize the sideeffects of chemotherapy. Examples of such agents include, but are notlimited to, amfostine, mesna, and dexrazoxane.

In one aspect of the invention, the subject compounds are administeredin combination with radiation therapy. Radiation is commonly deliveredinternally (implantation of radioactive material near cancer site) orexternally from a machine that employs photon (x-ray or gamma-ray) orparticle radiation. Where the combination therapy further comprisesradiation treatment, the radiation treatment may be conducted at anysuitable time so long as a beneficial effect from the co-action of thecombination of the therapeutic agents and radiation treatment isachieved. For example, in appropriate cases, the beneficial effect isstill achieved when the radiation treatment is temporally removed fromthe administration of the therapeutic agents, perhaps by days or evenweeks.

It will be appreciated that compounds of the invention can be used incombination with an immunotherapeutic agent. One form of immunotherapyis the generation of an active systemic tumor-specific immune responseof host origin by administering a vaccine composition at a site distantfrom the tumor. Various types of vaccines have been proposed, includingisolated tumor-antigen vaccines and anti-idiotype vaccines. Anotherapproach is to use tumor cells from the subject to be treated, or aderivative of such cells (reviewed by Schirrmacher et al. (1995) J.Cancer Res. Clin. Oncol. 121:487). In U.S. Pat. No. 5,484,596, Hanna Jr.et al. claim a method for treating a resectable carcinoma to preventrecurrence or metastases, comprising surgically removing the tumor,dispersing the cells with collagenase, irradiating the cells, andvaccinating the patient with at least three consecutive doses of about10⁷ cells.

It will be appreciated that the compounds of the invention mayadvantageously be used in conjunction with one or more adjunctivetherapeutic agents. Examples of suitable agents for adjunctive therapyinclude steroids, such as corticosteroids (amcinonide, betamethasone,betamethasone dipropionate, betamethasone valerate, budesonide,clobetasol, clobetasol acetate, clobetasol butyrate, clobetasol17-propionate, cortisone, deflazacort, desoximetasone, diflucortolonevalerate, dexamethasone, dexamethasone sodium phosphate, desonide,furoate, fluocinonide, fluocinolone acetonide, halcinonide,hydrocortisone, hydrocortisone butyrate, hydrocortisone sodiumsuccinate, hydrocortisone valerate, methyl prednisolone, mometasone,prednicarbate, prednisolone, triamcinolone, triamcinolone acetonide, andhalobetasol proprionate); a 5HTi agonist, such as a triptan (e.g.sumatriptan or naratriptan); an adenosine A1 agonist; an EP ligand; anNMDA modulator, such as a glycine antagonist; a sodium channel blocker(e.g. lamotrigine); a substance P antagonist (e.g. an NKi antagonist); acannabinoid; acetaminophen or phenacetin; a 5-lipoxygenase inhibitor; aleukotriene receptor antagonist; a DMARD (e.g. methotrexate); gabapentinand related compounds; a tricyclic antidepressant (e.g. amitryptilline);a neurone stabilising antiepileptic drug; a mono-aminergic uptakeinhibitor (e.g. venlafaxine); a matrix metalloproteinase inhibitor; anitric oxide synthase (NOS) inhibitor, such as an iNOS or an nNOSinhibitor; an inhibitor of the release, or action, of tumour necrosisfactor α; an antibody therapy, such as a monoclonal antibody therapy; anantiviral agent, such as a nucleoside inhibitor (e.g. lamivudine) or animmune system modulator (e.g. interferon); an opioid analgesic; a localanaesthetic; a stimulant, including caffeine; an H2-antagonist (e.g.ranitidine); a proton pump inhibitor (e.g. omeprazole); an antacid (e.g.aluminium or magnesium hydroxide; an antiflatulent (e.g. simethicone); adecongestant (e.g. phenylephrine, phenylpropanolamine, pseudoephedrine,oxymetazoline, epinephrine, naphazoline, xylometazoline,propylhexedrine, or levo-desoxyephedrine); an antitussive (e.g. codeine,hydrocodone, carmiphen, carbetapentane, or dextramethorphan); adiuretic; or a sedating or non-sedating antihistamine.

Methods for Inhibiting Expression of a Target Gene

In yet another aspect, the invention relates to a method for inhibitingthe expression of a target gene in a cell or organism. In oneembodiment, the method includes administering the inventiveoligonucleotide, e.g. antisense, aptamer, antagomir, or an iRNA agent;or a pharmaceutical composition containing the said oligonucleotide to acell or an organism, such as a mammal, such that expression of thetarget gene is silenced. Compositions and methods for inhibiting theexpression of a target gene using the inventive oligonucleotide, e.g. aniRNA agent, can be performed as described in the preceding sections.

In this embodiment, a pharmaceutical composition containing theinventive oligonucleotide may be administered by any means known in theart including, but not limited to oral or parenteral routes, includingintravenous, intramuscular, intraperitoneal, subcutaneous, transdermal,airway (aerosol), ocular, rectal, vaginal, and topical (including buccaland sublingual) administration. In preferred embodiments, thepharmaceutical compositions are administered by intravenous orintraparenteral infusion or injection. The pharmaceutical compositionscan also be administered intraparenchymally, intrathecally, and/or bystereotactic injection.

The methods for inhibiting the expression of a target gene can beapplied to any gene one wishes to silence, thereby specificallyinhibiting its expression, provided the cell or organism in which thetarget gene is expressed includes the cellular machinery which effectsRNA interference. Examples of genes which can be targeted for silencinginclude, without limitation, developmental genes including but notlimited to adhesion molecules, cyclin kinase inhibitors, Wnt familymembers, Pax family members, Winged helix family members, Hox familymembers, cytokines/lymphokines and their receptors,growth/differentiation factors and their receptors, andneurotransmitters and their receptors; (2) oncogenes including but notlimited to ABLI, BCL1, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2,ETS1, ETS1, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2,MLL, MYB, MYC, MYCL1, MYCN, NRAS, PIM1, PML, RET, SRC, TAL1, TCL3 andYES; (3) tumor suppresser genes including but not limited to APC, BRCA1,BRCA2, MADH4, MCC, NF1, NF2, RB1, TP53 and WT1; and (4) enzymesincluding but not limited to ACP desaturases and hydroxylases,ADP-glucose pyrophorylases, ATPases, alcohol dehydrogenases, amylases,amyloglucosidases, catalases, cellulases, cyclooxygenases,decarboxylases, dextrinases, DNA and RNA polymerases, galactosidases,glucanases, glucose oxidases, GTPases, helicases, hemicellulases,integrases, invertases, isomerases, kinases, lactases, lipases,lipoxygenases, lysozymes, pectinesterases, peroxidases, phosphatases,phospholipases, phosphorylases, polygalacturonases, proteinases andpeptidases, pullanases, recombinases, reverse transcriptases,topoisomerases, and xylanases.

In addition to in vivo gene inhibition, the skilled artisan willappreciate that the inventive oligonucleotides, e.g. iRNA agent, of thepresent invention are useful in a wide variety of in vitro applications.Such in vitro applications, include, for example, scientific andcommercial research (e.g., elucidation of physiological pathways, drugdiscovery and development), and medical and veterinary diagnostics. Ingeneral, the method involves the introduction of the oligonucleotide,e.g. an iRNA agent, into a cell using known techniques (e.g., absorptionthrough cellular processes, or by auxiliary agents or devices, such aselectroporation and lipofection), then maintaining the cell for a timesufficient to obtain degradation of an mRNA transcript of the targetgene.

DEFINITIONS

The term “aliphatic,” as used herein, refers to a straight or branchedhydrocarbon radical containing up to twenty four carbon atoms whereinthe saturation between any two carbon atoms is a single, double ortriple bond. An aliphatic group preferably contains from 1 to about 24carbon atoms, more typically from 1 to about 12 carbon atoms. Suitablealiphatic groups include, but are not limited to, linear or branched,substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybridsthereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or(cycloalkyl)alkenyl. The straight or branched chain of an aliphaticgroup may be interrupted with one or more heteroatoms that includenitrogen, oxygen, sulfur and phosphorus. Such aliphatic groupsinterrupted by heteroatoms include without limitation polyalkoxys, suchas polyalkylene glycols, polyamines, and polyimines, for example.Aliphatic groups as used herein may optionally include furthersubstitutent groups.

The term “alkyl” refers to saturated and unsaturated non-aromatichydrocarbon chains that may be a straight chain or branched chain,containing the indicated number of carbon atoms (these include withoutlimitation propyl, allyl, or propargyl), which may be optionallyinserted with N, O, or S. For example, C₁-C₂₀ indicates that the groupmay have from 1 to 20 (inclusive) carbon atoms in it. The term “alkoxy”refers to an —O-alkyl radical. The term “alkylene” refers to a divalentalkyl (i.e., —R—). The term “alkylenedioxo” refers to a divalent speciesof the structure —O—R—O—, in which R represents an alkylene. The term“aminoalkyl” refers to an alkyl substituted with an amino. The term“mercapto” refers to an —SH radical. The term “thioalkoxy” refers to an—S-alkyl radical.

The term “cyclic” as used herein includes a cycloalkyl group and aheterocyclic group. Any suitable ring position of the cyclic group maybe covalently linked to the defined chemical structure.

The term “acyclic” may describe any carrier that is branched orunbranched, and does not form a closed ring.

The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclicaromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring may besubstituted by a substituent. Examples of aryl groups include phenyl,naphthyl and the like. The term “arylalkyl” or the term “aralkyl” refersto alkyl substituted with an aryl. The term “arylalkoxy” refers to analkoxy substituted with aryl.

The term “cycloalkyl” as employed herein includes saturated andpartially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons,for example, 3 to 8 carbons, and, for example, 3 to 6 carbons, whereinthe cycloalkyl group additionally may be optionally substituted.Cycloalkyl groups include, without limitation, decalin, cyclopropyl,cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl,cycloheptyl, and cyclooctyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3,or 4 atoms of each ring may be substituted by a substituent. Examples ofheteroaryl groups include pyridyl, furyl or furanyl, imidazolyl,benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl,thiazolyl, and the like. The term “heteroarylalkyl” or the term“heteroaralkyl” refers to an alkyl substituted with a heteroaryl. Theterm “heteroarylalkoxy” refers to an alkoxy substituted with heteroaryl.

The term “heterocycloalkyl” and “heterocyclic” can be usedinterchangeably and refer to a non-aromatic 3-, 4-, 5-, 6- or 7-memberedring or a bi- or tri-cyclic group fused system, where (i) each ringcontains between one and three heteroatoms independently selected fromoxygen, sulfur and nitrogen, (ii) each 5-membered ring has 0 to 1 doublebonds and each 6-membered ring has 0 to 2 double bonds, (iii) thenitrogen and sulfur heteroatoms may optionally be oxidized, (iv) thenitrogen heteroatom may optionally be quaternized, (iv) any of the aboverings may be fused to a benzene ring, and (v) the remaining ring atomsare carbon atoms which may be optionally oxo-substituted. Representativeheterocycloalkyl groups include, but are not limited to, [1,3]dioxolane,pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl,piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl,thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, andtetrahydrofuryl. Such heterocyclic groups may be further substituted togive substituted heterocyclic.

The term “oxo” refers to an oxygen atom, which forms a carbonyl whenattached to carbon, an N-oxide when attached to nitrogen, and asulfoxide or sulfone when attached to sulfur.

The term “acyl” refers to an alkylcarbonyl, cycloalkylcarbonyl,arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent,any of which may be further substituted by substituents.

The term “silyl” as used herein is represented by the formula —SiA¹A²A³,where A¹, A², and A³ can be, independently, hydrogen or a substituted orunsubstituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl,cycloalkynyl, aryl, or heteroaryl group as described herein.

The term “substituted” refers to the replacement of one or more hydrogenradicals in a given structure with the radical of a specifiedsubstituent including, but not limited to: halo, alkyl, alkenyl,alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alkylthioalkyl,arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl,alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl,arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino,trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl,arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl,alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl,carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl,heteroaryl, heterocyclic, and aliphatic. It is understood that thesubstituent may be further substituted.

The Bases

Adenine, guanine, cytosine and uracil are the most common bases found inRNA. These bases can be modified or replaced to provide RNA's havingimproved properties. E.g., nuclease resistant oligoribonucleotides canbe prepared with these bases or with synthetic and natural nucleobases(e.g., inosine, thymine, xanthine, hypoxanthine, nubularine,isoguanisine, or tubercidine) and any one of the above modifications.Alternatively, substituted or modified analogs of any of the above basesand “universal bases” can be employed. Examples include 2-aminoadenine,2-fluoroadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo,amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines andguanines, 5-trifluoromethyl and other 5-substituted uracils andcytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidinesand N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine,dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil,7-alkylguanine, 5-alkyl cytosine, 7-deazaadenine, N6,N6-dimethyladenine,2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole,5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil,5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil,5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil,3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine,N6-methyladenine, N6-isopentyladenine,2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylatedbases. Further purines and pyrimidines include those disclosed in U.S.Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia OfPolymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed.John Wiley & Sons, 1990, and those disclosed by Englisch et al.,Angewandte Chemie, International Edition, 1991, 30, 613.

The term “non-natural” nucleobase refers any one of the following:2-methyladeninyl, N6-methyladeninyl, 2-methylthio-N6-methyladeninyl,N6-isopentenyladeninyl, 2-methylthio-N6-isopentenyladeninyl,N6-(cis-hydroxyisopentenyl)adeninyl,2-methylthio-N6-(cis-hydroxyisopentenyl) adeninyl,N6-glycinylcarbamoyladeninyl, N6-threonylcarbamoyladeninyl,2-methylthio-N6-threonyl carbamoyladeninyl,N6-methyl-N6-threonylcarbamoyladeninyl,N6-hydroxynorvalylcarbamoyladeninyl, 2-methylthio-N6-hydroxynorvalylcarbamoyladeninyl, N6,N6-dimethyladeninyl, 3-methylcytosinyl,5-methylcytosinyl, 2-thiocytosinyl, 5-formylcytosinyl,N4-methylcytosinyl, 5-hydroxymethylcytosinyl, 1-methylguaninyl,N2-methylguaninyl, 7-methylguaninyl, N2,N2-dimethylguaninyl,N2,7-dimethylguaninyl, N2,N2,7-trimethylguaninyl, 1-methylguaninyl,7-cyano-7-deazaguaninyl, 7-aminomethyl-7-deazaguaninyl, pseudouracilyl,dihydrouracilyl, 5-methyluracilyl, 1-methylpseudouracilyl,2-thiouracilyl, 4-thiouracilyl, 2-thiothyminyl 5-methyl-2-thiouracilyl,3-(3-amino-3-carboxypropyl)uracilyl, 5-hydroxyuracilyl,5-methoxyuracilyl, uracilyl 5-oxyacetic acid, uracilyl 5-oxyacetic acidmethyl ester, 5-(carboxyhydroxymethyl)uracilyl,5-(carboxyhydroxymethyOuracilyl methyl ester,5-methoxycarbonylmethyluracilyl, 5-methoxycarbonylmethyl-2-thiouracilyl,5-aminomethyl-2-thiouracilyl, 5-methylaminomethyluracilyl,5-methylaminomethyl-2-thiouracilyl,5-methylaminomethyl-2-selenouracilyl, 5-carbamoylmethyluracilyl,5-carboxymethylaminomethyluracilyl,5-carboxymethylaminomethyl-2-thiouracilyl, 3-methyluracilyl,1-methyl-3-(3-amino-3-carboxypropyl) pseudouracilyl,5-carboxymethyluracilyl, 5-methyldihydrouracilyl,3-methylpseudouracilyl,

A universal base can form base pairs with each of the natural DNA/RNAbases, exhibiting relatively little discrimination between them. Ingeneral, the universal bases are non-hydrogen bonding, hydrophobic,aromatic moieties which can stabilize e.g., duplex RNA or RNA-likemolecules, via stacking interactions. A universal base can also includehydrogen bonding substituents. As used herein, a “universal base” caninclude anthracenes, pyrenes or any one of the following:

Antagomirs

Antagomirs are RNA-like oligonucleotides that harbor variousmodifications for RNAse protection and pharmacologic properties, such asenhanced tissue and cellular uptake. They differ from normal RNA by, forexample, complete 2′-O-methylation of sugar, phosphorothioate backboneand, for example, a cholesterol-moiety at 3′-end. Antagomirs may be usedto efficiently silence endogenous miRNAs thereby preventingmiRNA-induced gene silencing. An example of antagomir-mediated miRNAsilencing is the silencing of miR-122, described in Krutzfeldt et al,Nature, 2005, 438: 685-689, which is expressly incorporated by referenceherein, in its entirety.

Decoy Oligonucleotides

Because transcription factors can recognize their relatively shortbinding sequences, even in the absence of surrounding genomic DNA, shortoligonucleotides bearing the consensus binding sequence of a specifictranscription factor can be used as tools for manipulating geneexpression in living cells. This strategy involves the intracellulardelivery of such “decoy oligonucleotides”, which are then recognized andbound by the target factor. Occupation of the transcription factor'sDNA-binding site by the decoy renders the transcription factor incapableof subsequently binding to the promoter regions of target genes. Decoyscan be used as therapeutic agents, either to inhibit the expression ofgenes that are activated by a transcription factor, or to upregulategenes that are suppressed by the binding of a transcription factor.Examples of the utilization of decoy oligonucleotides may be found inMann et al., J. Clin. Invest., 2000, 106: 1071-1075, which is expresslyincorporated by reference herein, in its entirety.

Antisense Oligonucleotides

Antisense oligonucleotides are single strands of DNA or RNA that are atleast partially complementary to a chosen sequence. In the case ofantisense RNA, they prevent translation of complementary RNA strands bybinding to it. Antisense DNA can also be used to target a specific,complementary (coding or non-coding) RNA. If binding takes place, theDNA/RNA hybrid can be degraded by the enzyme RNase H. Examples of theutilization of antisense oligonucleotides may be found in Dias et al.,Mol. Cancer Ther., 2002, 1: 347-355, which is expressly incorporated byreference herein, in its entirety.

Aptamers

Aptamers are nucleic acid molecules that bind a specific target moleculeor molecules. Aptamers may be RNA or DNA based, and may include ariboswitch. A riboswitch is a part of an mRNA molecule that can directlybind a small target molecule, and whose binding of the target affectsthe gene's activity. Thus, an mRNA that contains a riboswitch isdirectly involved in regulating its own activity, depending on thepresence or absence of its target molecule.

REFERENCES

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. It is, therefore, to beunderstood that the foregoing embodiments are presented by way ofexample only and that, within the scope of the appended claims andequivalents thereto, the invention may be practiced otherwise than asspecifically described and claimed.

EXAMPLES

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the invention, and are not intended to limit the invention. Thus, theinvention should in way be construed as being limited to the followingexamples, but rather, should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein.

Example 1 Synthesis of Lipidic Azides

Docosyl Azide (1).

Docosyl bromide (10.0 g, 25.67 mmol) in anhydrous DMF (100 ml) wastreated with sodium azide (8.35 g, 128.37 mmol) at 60° C. for 2 h. Thendilution of the reaction solution with ice water (1000 ml) gave whitecrystal. Filtration and drying on suction funnel and vacuum gave 1 as awhite crystal (9.11 g, 25.90 mmol, quant). LC-MS calcd for [M+Na]⁺621.2. found 621.2.

Stearyl Azide (2).

Bromooctadecane (10.0 g, 30.10 mmol) in anhydrous DMF (200 ml) wastreated with sodium azide (9.78 g, 150.51 mmol) at 60° C. for 2 h. Thendilution of the reaction solution with ice water gave white crystal.Filtration and drying on suction funnel and then vacuum gave 2 as awhite crystal. (6.82 g, 23.07 mmol, 76%)

Linoleyl Azide (3).

Linoleyl alcohol (10 ml, 32.3 mmol) in anhydrous DCM (80 ml) was treatedwith methanesulfonylchloride (2.75 ml, 35.5 mmol) and triethylamine (5.8ml, 42.0 mmol) at room temperature for 2 h. Extraction with DCM/water,drying organic layer with sodium sulfate, and then evaporation gavecrude mesylate. This crude was directly used for next step.

The crude in anhydrous DMF (300 ml) was treated with sodium azide (10.5g, 161.50 mmol) at 60° C. for 18 h. Extraction with ethylacetate/water,drying organic layer with sodium sulfate, and then evaporation gave darkoil. The oil was chromatographed on silica gel. Eluent with Hexane gave3 as clear oil (7.51 g, 25.67 mmol, 80% (2 steps)).

Oleyl Azide (4).

Oleyl alcohol (10 ml, 32.30 mmol) in anhydrous DCM (80 ml) was treatedwith methanesulfonylchloride (2.75 ml, 35.5 mmol) and triethylamine (5.8ml, 42.0 mmol) at room temperature for 4 h. Extraction with DCM/water,drying organic layer with sodium sulfate, and then evaporation gavecrude mesylate. This crude was directly used for next step.

The crude in anhydrous DMF (300 ml) was treated with sodium azide (10.5g, 161.50 mmol) at 60° C. for 18 h. Extraction with ethylacetate/water,drying organic layer with sodium sulfate, and then evaporation gave darkoil. The oil was chromatographed on silica gel. Eluent with Hexane gave3 as clear oil (4.00 g, 16.38 mmol, 44% (2 steps)).

Hydroxyhexylcarbamoylcholesterol (5).

1-amino-6-hexanol (5.00 g, 42.67 mmol) in anhydrous dichloromethane wastreated with triethylamine (11.9 ml, 85.33 mmol) and cholesterolchloroformate (19.16 g, 42.67 mmol) at room temperature for 4 h.Extraction with DCM/brine, drying with sodium sulfate and evaporationgave yellow syrup. Then crystallized with DCM/Hexane gave 5 as whitecrystal (20.3 g, 38.31 mmol, 89%). LC-MS calcd for [M+Na]⁺ 552.3. found552.3.

Azidohexylcarbamoylcholesterol (6).

5 (15.0 g, 28.31 mmol) was treated with triethylamine (7.9 ml, 56.62mmol) and methanesulfonylchloride (2.4 ml, 31.14 mmol) at roomtemperature for 4 h. Extraction with DCM/brine, drying, concentration invacuo gave crude mesylate. This crude was directly used next step.

The crude in anhydrous DMF (300 ml) was treated with sodium azide (300ml) at 60° C. for 18 h. Then Extraction with ethyl acetate/brine, dryingorganic layer with sodium sulfate and evaporation gave yellow syrup. Thesyrup was crystallized after the syrup was still stood for 18 h. Thecrystal was washed with water. Filtration and drying on a suction funneland then vacuum gave 6 as a yellow crystal (13.24 g, 23.87 mmol, 84% (2steps)).

Example 2 Lipid Alkyne Conjugate

General Procedure.

Each carbonyl acid or chloroformate in DMF is treated with HBTU andpropargyl amine to give corresponding alkynes.

Example 3 Lipid Azide Ester Conjugate

These azides will be used for post-synthesis after NH₄OH treatment.

Ester will be cleaved with esterase in a cell.

General Procedure.

Each Chloride or chloroformate in DCM are treated with TEA (2 equiv) and1-bromohexanol (1.1 equiv) at room temperature. Aqueous work-up andconcentration give crude bromide. The bromide in DMF is treated withsodium azide (5 equiv) at 60° C. Recrystallization gives correspondingazide.

Example 4 Synthesis of 2′/3′-O-propargylnucleoside derivatives

Compound 100b.

5-methyluridine (20.0 g, 77.45 mmol) in pyridine (350 ml) was treatedwith chlorotrimethylsilane (49.4 ml, 42.07 mmol) for 30 min at roomtemperature. To the solution was added benzoyl chloride (9.90 ml, 11.98mmol) in an ice bath. After 18 h, TLC monitoring and LC-MS analysisshowed reaction completed. To the solution was added ice water (100 ml)in an ice bath. After 2 h, TLC monitoring and LC-MS analysis showed thecleavage of three trimethylsilyl ethers. The solution was directlyextracted with ethyl acetate and brine. The organic layer was dried withsodium sulfate and then concentrated under reduced pressure. The residuewas chromatographed on silica gel. Eluent with 5% MeOH in DCM gave 100aas white foam (23.5 g, 64.36 mmol, 82%). LC-MS calcd for [M+Na]⁺ 385.1.found 385.1.

Compound 101b and 102b.

To the solution of 100a (330 mg, 0.91 mmol) in anhydrous DMF was addeddibutyltinoxide (249 mg, 1.00 mmol) at 100° C. After 18 h, reaction wasquenched with water. The reaction solution was concentrated underreduced pressure and then chromatographed on silica gel without aqueouswork-up. Eluent with 10% MeOH in DCM gave mixture of 101b and 102b (140mg, 0.34 mmol, 38%) as foam.

Compound 103b.

To the solution of 101b and 102b (5.00 g, 12.48 mmol) in anhydrouspyridine (200 ml) was added 4,4′-dimethoxytritylchloride (6.00 g, 17.71mmol). After 4 h, TLC monitoring showed reaction completed. Then Thesolution was extracted with EtOAc and brine after quenched with water.The organic layer was concentrated under reduced pressure to give yellowresidue. The residue was treated with 7 M ammonia in MeOH for 18 h atroom temperature. Then the solution was evaporated to give yellowresidue. The residue was chromatographed on silica gel. Eluent withEtOAc/Hexane (1:1 to 1:2) gave 103b as white foam (3.165 g, 5.41 mmol,30%). LC-MS calcd for [M+Na]⁺ 621.2. found 621.2.

Compound 104b.

104b was eluted after 103b was eluted. Yield was 770 mg (1.31 mmol, 8%).LC-MS calcd for [M+Na]⁺ 621.2. found 621.2.

Compound 105a.

To the solution of 5′-O-DMTruridine (9.00 g, 36.75 mmol) in anhydrousDMF was added sodium hydride (1.94 g, 81.08 mmol) in an ice bath. Aftervigorous stirring for 1 h, chloromethyl pivalate (11.85 ml, 81.08 mmol)was added to the solution in an ice bath. After vigorous stirring for 24h at 0° C. to room temperature, TLC monitoring showed reaction complete.The solution was partitioned with EtOAc and aqueous ammonium chloridesolution. The organic layer was dried with sodium sulfate andconcentrated under reduced pressure to give yellow residue.Chromatography gives 105a.

Compound 105b.

Similar procedure to 105a gives 105b.

Compound 106a.

To the solution of 105a in benzene/hexane (4:1, v/v) is addeddibutyltinoxide (1.1 equiv). The mixture is irradiated by microwave at120° C. for 20 min. After reaction, the mixture turns into clearsolution. Then propargyl bromide (5 equiv) and tetrabutylammonium iodide(5 equiv) are added to the solution. The solution was irradiated bymicrowave at 120° C. for 2.5 h. Aqueous work-up and chromatography give106a.

Compound 106b.

Similar procedure to 106a gives 106b.

Compound 107a.

107a is eluted after 106a.

Compound 107b.

Similar procedure to 107a gives 107b.

Example 5 Synthesis of 5′-O-propargylcarbamoylnucleoside derivatives

Compound 108a.

2′-3′-O-Isopropyrideneuridine (10.00 g, 35.18 mmol) in pyridine (350 ml)was treated with phenylchloroformate (4.8 ml, 38.70 mmol) at roomtemperature for 2 h. Then propargylamine (7.3 ml, 175.89 mmol) was addedto the solution. After vigorous stirring 18 h at room temperature,TLC-monitoring showed reaction completed. The solution was directlypartitioned with DCM and sat NaHCO₃aq. The organic layer was dried withsodium sulfate and concentrated under reduced pressure to give darkresidue. The residue was chromatographed on silica gel. Eluent with 2%MeOH in DCM gave 108a as white foam (11.73 g, 32.10 mmol, 91% (2 steps).LC-MS calcd for [M+H]⁺ 366.0. found 366.0.

Compound 108b.

Similar procedure to 108a gives 108b.

Compound 109a.

108a (11.73 g, 32.10 mmol) in MeOH (20 ml) was refluxed with 80% AcOH(200 ml) for 18 h. Completion of the reaction was checked by LC-MS. Thereaction solution was concentrated under reduced pressure to give yellowgummy syrup. The syrup was treated with MeOH (50 ml) and then sonicatedfor 30 min Sonication gave white crystal. Filtration and drying onsuction funnel and then vacuum gave 109a as white crystal (10.34 g,31.78 mmol, 99%).

Compound 109b.

Similar procedure to 109a gives 109b.

Compound 110a.

109a (10.34 g, 28.30 mmol) in anhydrous THF (30 ml) was treated withsilver nitrate (5.77 g, 34.00 mmol), pyridine (17.0 ml, 209.4 mmol) andtertiallybutyldimethylsilyl chloride (4.27 g, 28.30 mmol) at roomtemperature for 18 h. Then the solution was partitioned with DCM/water.The organic layer was dried with sodium sulfate and then concentratedunder reduced pressure to give residue. The residue was chromatographedon silica gel. 110a and 3′-O-isomer (4.00 g, 32%) was eluted withHexane/EtOAc (1:1) at the same time. Then the mixture waschromatographed on aluminum gel. Eluent with 2% MeOH in DCM gave pure110a. LC-MS calcd for [M+Na]⁺ 462.1. found 462.1.

Compound 110b.

Similar procedure to 110a gives 110b.

Compound 111a.

110a (452 mg, 1.02 mmol) in anhydrous DCM (12 ml) was treated withdiisopropylethylamine (612 μl, 3.41 mmol) and2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (380 ml, 1.71 mmol) atroom temperature for 6 h. The solution was extracted with DCM/brine.Then the organic layer was dried with sodium sulfate and concentratedunder reduced pressure to give yellow residue. The residue waschromatographed on silica gel. Eluent with TEA/Hexane/EtOAc (0.5:50:50)gave 111a (469 mg, 0.73 mmol, 71%) as white foam.

Compound 111b.

Similar procedure to 111a gives 111b.

Example 6 ‘Clicked’ Compounds by Using of Propargylnulceosides andAzides

Compound 112a.

103b (25 mg, 0.04 mmol) in DCM/MeOH (4:1, v/v) (0.5 ml) was treated with1 (14 mg, 0.04 mmol), tetrakisacetonitrilecopper hexafluorophosphate (3mg, 0.01 mmol) and copper (1 mg, 0.01 mmol). After 1 h, the solution wasextracted with DCM/brine. The organic layer was concentrated underreduced pressure to give green residue. The residue was chromatographedon silica gel. Eluent with 1% MeOH in DCM gave 112a as white foam (36mg, 0.037 mmol, 97%). LC-MS calcd for [M+Na]⁺ 972.5. found 972.5.

Compound 113.

Typical phosphytilation gives 113.

Compound 114.

Typical succinilation gives 114.

Compound 115.

Typical immobilization to CPG gives 115.

Compound 116.

Similar procedure to 112a gives 116.

Compound 117.

Typical phosphytilation gives 117.

Compound 118.

Typical succinilation gives 118.

Compound 119.

Typical immobilization to CPG gives 119.

Compound 120a.

110a (100 mg, 0.17 mmol) in DCM/MeOH (4:1, v/v) (2 ml) was treated with3 (59 mg, 0.17 mmol), tetrakisacetonitrilecopper hexafluorophosphate (13mg, 0.03 mmol) and copper (2 mg, 0.03 mmol) at room temperature for 3 h.The solution was concentrated under reduced pressure and chromatographedon silica gel without aqueous work-up. Eluent with hexane/EtOAc (1:3)gave 120a as foam (122 mg, 0.16 mmol, 98%). LC-MS calcd for [M+H]⁺731.2. found 731.2.

Compound 120b.

110a (500 mg, 1.14 mmol) in DCM/MeOH (4:1, v/v) was treated with 325(460 mg, 1.14 mmol), tetrakisacetonitrilecopper hexafluorophosphate (42mg, 0.11 mmol) and copper (7 mg, 0.11 mmol) at room temperature for 18h. Then the solution was concentrated under reduced pressure andchromatographed on silica gel directly without aqueous work-up. Eluentwith DCM/MeOH (10:1) gave 120b as white foam (800 mg, 0.88 mmol, 88%).

Compound 121a.

120a (730 mg, 1.00 mmol) in anhydrous DCM (10 ml) was treated withdiisopropylethylamine (525 μl, 3.00 mmol) and2-cyanoethyl-N,N-diisopropylethylphosphoramidite (335 μl, 1.50 mmol) atroom temperature for 4 h. The solution was then extracted withDCM/brine. The organic layer was dried with sodium sulfate and thenconcentrated under reduced pressure to give dark residue. The residuewas chromatographed on silica gel. Eluent with TEA/Hexane/EtOAc(0.5:60:30) gave 116a as white foam (463 mg, 0.50 mmol, 50%).

Compound 121b.

120b (400 g, 0.44 mmol) in anhydrous DCM (4 ml) was treated withdiisopropylethylamine (230 μl, 1.33 mmol) and2-cyanoethyl-N,N-diisopropylethylphosphoramidite (150 μl, 0.67 mmol) atroom temperature for 6 h. The solution was partitioned with DCM/brine.The organic layer was dried with sodium sulfate and then concentratedunder reduced pressure to give yellow residue. The residue waschromatographed on silica gel. Eluent with TEA/MeOH/EtOAc (0.5:2:100)gave 120b as white foam (403 mg, 0.36 mmol, 83%). LC-MS calcd for[M+Na]⁺ 1123.3. found 1123.3.

Example 7 Sugar Ligands with Functionalized Alkynes or Azides

Preparation of 319:

Galactosamine pentaacetate 300 (52.00 g, 133.63 mmol) was taken indichloroethane (300 mL) at ambient temperature. TMSOTf (44.55 g, 200.44mmol) was added that and the mixture stirred at 50° C. for 90 minutes ina water bath, heating stopped and the mixture stirred overnight at roomtemperature. It was poured in to an ice cold sodium bicarbonatesolution; extracted with dichloromethane, washed with water and driedover sodium sulfate. Solvents were removed in vacuo and the residue wasdried under high vacuum overnight to get the compound 301 as a dark gum(TLC: 15% MeOH in CHCl₃, Rf Value: 0.5, 44.50 g, quantitative). It wasused for next reaction with out further purification. ¹H NMR and MALDIconfirmed the product formation. ¹H NMR (CDCl₃ 400 MHz) δ□=6.01 (d,J=6.8 Hz, 1H), 5.46 (t, J=3.01 Hz, 1H), 4.91 (dd, J=3.29, 7.30 Hz, 1H),4.26-3.93 (m, 4H), 2.12 (s, 3H), 2.07 (s, 6H), 2.05 (s, 3H). MS: MWcalc. for C₁₄H₁₉NO₈: 329.11. Found 352.1 (M+Na).

Preparation of 303:

Compound 301 (43.70 g, 133.56 mmol) and the benzyl ester 302 (41.71 g,200.34 mmol) were dissolved in dichloroethane (300 mL), molecular sieves(50 g) was added to the solution and stirred for 30 minutes. TMSOTf(14.50 g, 66.78 mmol) was added and the mixture was stirred overnight atroom temperature. It was poured into an ice cold solution of sodiumbicarbonate and the product was extracted into dichloromethane, washedwith water and dried over anhydrous sodium sulfate. Solvents wereremoved in vacuo and the residue was purified by silica gel columnchromatography (gradient elution: 20-100% ethylacetate/hexanes) to getthe required compound 303 as light brown gummy liquid (60.50 g, 86%).¹HNMR (DMSO-d₆ 400 MHz) δ=7.76 (d, J-9.03 Hz, 1H), 7.25-7.38 (m, 5H),5.20 (d, J=3.17 Hz, 1H), 5.05 (s, 2H), 4.95 (dd, J=3.41, 11.23 Hz, 1H),4.48 (d, J=8.30 Hz, 1H), 3.70-4.20 (m, 4H), 3.35-3.45 (m, 2H), 2.33 (t,J=7.32 Hz, 2H), 2.08 (s, 3H), 1.97 (s, 3H), 1.87 (s, 3H), 1.73 (s, 3H),1.50-1.63 (m, 4H). MS: MW Calc. for C₂₆H₃₅NO₁₁: 537.22. Found 560.21(M+Na).

Preparation 304:

Compound 303 (60.00 g, 111.68 mmol) was dissolved in a mixture ofMethanol/ethylacetate and degassed with argon. Pd/C (6.00 g, 10 wt %Degussa, wet type) was added and hydrogenated under balloon pressureovernight. Filtered through a small pad of celite; washed with methanoland dried under high vacuum overnight to get the acid (48.85 g, 98%).¹HNMR (DMSO-d₆ 400 MHz) δ=11.97 (bs, 1H), 7.78 (d, J-9.20 Hz, 1H), 5.21(d, J=3.16 Hz, 1H), 4.96 (dd, J=3.42, 11.30 Hz, 1H), 4.47 (d, J=8.26 Hz,1H), 3.73-4.22 (m, 4H), 3.32-3.45 (m, 2H), 2.09 (s, 3H), 1.98 (s, 3H),1.87 (s, 3H), 1.75 (s, 3H), 1.51-1.64 (m, 4H) MS: MW Calc. forC₁₉H₂₉NO₁₁: 447.17. Found 469.9 (M+Na). Part of the acid (5 g, 11.17mmol), Propargyl amine (0.690 g, 12.29 mmol) and HBTU (4.23 g, 11.17mmol) are dissolved in DMF (50 mL). DIEA is added to the reactionmixture and stir the reaction mixture overnight. Remove the solventunder reduced pressure and the residue extracted with dichloromethane,washed with water and dried over anhydrous sodium sulfate. Solvents areremoved in vacuo and the residue purify by silica gel chromatography toget the product 304. MS: MW Calc. for C₂₂H₃₂NO₁₀: 484.21.

Preparation of 305:

Compound 304 (3.00 g, 6.19 mmol) is dissolved in a mixture ofDCM/MeOH(1:2, 20 mL) and sodium methoxide solution (20 mL, 0.5 M inMeOH) is added to that and stir the mixture overnight. Neutralize thereaction mixture with AcOH and remove the solvents under reducedpressure. Residue is purified by silica gel chromatography to get therequired product 305. MS: MW Calc. for C₁₆H₂₆NO₇: 358.39

Preparation of 307:

Compound 301 (42.30 g, 128.43 mmol) and the azido ethanol 306 (26 g,192.45 mmol) were dissolved in dichloroethane (300 mL), molecular sieves(50 g) was added to the solution and stirred for 30 minutes. TMSOTf(14.29 g, 64.21 mmol) was added and the mixture was stirred overnight atroom temperature. It was poured into an ice cold solution of sodiumbicarbonate and the product was extracted into dichloromethane, washedwith water and dried over anhydrous sodium sulfate. Solvents wereremoved in vacuo and the residue was purified by silica gel columnchromatography (gradient elution: 20-100% ethyl acetate/hexanes,followed by 5-10% Methanol/ethyl acetate) to obtain the requiredcompound 307 as light brown gummy liquid (59.23 g, 91.00%). ¹HNMR(DMSO-d₆ 400 MHz) δ=7.77 (d, J-9.27 Hz, 1H), 5.20 (d, J=3.15 Hz, 1H),4.97 (dd, J=3.39, 11.34 Hz, 1H), 4.55 (d, J=8.64 Hz, 1H), 3.72-4.22 (m,5H), 3.30-3.63 (m, 11H), 2.09 (s, 3H), 1.98 (s, 3H), 1.88 (s, 3H), 1.76(s, 3H), MS: MW Calc. for C₂₀H₃₂N₄O₁₁: 504.21. Found 527.1 (M+Na).

Preparation of 308:

Compound 307 (5.85 g, 11.59 mmol) was dissolved in a mixture ofDCM/MeOH(1:2, 50 mL) and sodium methoxide solution (20 mL, 0.5 M inMeOH) was added to that and stir the mixture overnight. Neutralize thereaction mixture with AcOH and removed the solvents under reducedpressure. Residue was purified by silica gel chromatography (DCM, 20%MeOH/DCM) to get the required product 308. MS: MW Calc. for C₁₄H₂₆N₄O₈:378.18. Found 401.19 (M+Na).

Preparation of 311:

PEG acid 310 (5.50 g, 2.908 mmol) was dissolved in DMF (50 mL); TBTU(1.23 g, 3.83 mmol), HOBt (0.520 g, 3.83 mmol) and DIEA (3.2 mL, 5 eq)were added to the mixture and stirred for 3-4 minutes. A solution of 309(1.02 g, 1.2 eq) in DMF was added to the mixture and stirred thereaction mixture overnight. TLC was checked, solvents were removed underreduced pressure. The residue was dissolved in dichloromethane, washedwith sodium bicarbonate solution (50 mL), water (50 mL) and dried overanhydrous sodium sulfate. Solvents were removed in vacuo and the residuewas purified by silica gel column chromatography (ethyl acetate,followed by gradient elution 5-15% MeOH/DCM) to get the product 311 asan off white solid (3.30 g, 56%). MS: MW Calc. for C₉₀H₁₄₇N₁₃O₄₁:2065.98. Found 2089.09 (M+Na).

Preparation of 312:

Compound 311 (0.92 g, 0.445 mmol) was dissolved in MeOH(10 mL) andsodium methoxide solution (10 mL, 0.5 M in MeOH) was added to that andstir the mixture overnight. Reaction was monitored by TLC and themixture passed through a column of CM Sepharose resin. Washed with MeOHand removed the solvents in vacuo to get the product 312 (0.42 g, 57%).MS: MW Calc. for C₇₂H1₂₉N₁₃O₃₂: 1687.89. Found 1710.90 (M+Na).

Preparation of 313:

Compound 311 (2.00 g, 0.967 mmol) is dissolved in THF (20 mL) to thatPPh₃ (0.380 g, 1.45 mmol) is added and the mixture stir for 48 h. TLC ischecked to see complete disappearance of starting material. Water (0.1mL, 5 eq) is added to the reaction mixture and stir for another 24 h.TFA (0.140 g, 1.20 mmol) and toluene (30 mL) are added and theconcentrate under reduced pressure. The residue co-evaporates withtoluene (2×30 mL) twice and dries under high vacuum. The product thusobtained can be used for the next reaction in the same day withoutfurther purification. MS: MW Calc. for C₉₀H₁₄₉N₁₁O₄₁: 2039.99. Compoundfrom the above reaction and propynoic acid (0.081 g, 1.2 eq) aredissolved in DMF (20 mL). To that add HBTU (0.440 g, 1.16 mmol) andDIEA, stir the mixture overnight. Solvents are removed under reducedpressure. The residue is dissolved in dichloromethane, wash with sodiumbicarbonate solution, water and dry over anhydrous sodium sulfate.Solvents are removed in vacuo and the residue is purified by silica gelcolumn chromatography to get the product 313.

Preparation of 314:

Compound 311 (1.00 g, 0.447 mmol) is dissolved in MeOH(10 mL) and sodiummethoxide solution (10 mL, 0.5 M in MeOH) is added to that and stir themixture overnight. Reaction is monitored by TLC and the mixtures passthrough a column of CM Sepharose resin. Wash with MeOH and remove thesolvents in vacuo to get the product 314.

Preparation of 316:

Galactose trichloroacetimidate 315 (10.00 g, 13.53 mmol) and the benzylester 302 (3.90 g, 14.88 mmol) are co evaporated with toluene (2×40 ml)two times and dried under high vacuum. Anhydrous ether (50 mL) andmolecular sieves (20 g) are added to the solution and cool in an icebath. TMSOTf (0.300 g, 1.349 mmol) is added and the mixtures stir for 15minutes. Solvent is removed and purify the residue by silica gel columnchromatography (gradient elution: 20-60% ethyl acetate/hexanes) to getthe required compound 316.

Preparation of 317:

Compound 316 (5.00 g, 6.35 mmol) is taken in MeOH and hydrogenated underballoon pressure using Pd/C to get the acid. Crude acid and propargylamine (0.384 g, 6.99 mmol) and HBTU (2.40 g, 6.35 mmol) are dissolved inDMF (50 mL). DIEA is added and stir the reaction mixture overnight.Solvent is removed and the residue is purified by silica gelchromatography to get the required product 317.

Preparation of 318:

Compound 317 (2.00 g, 2.72 mmol) is dissolved in a mixture ofDCM/MeOH(1:2, 50 mL) and sodium methoxide solution (20 mL, 0.5 M inMeOH) is added to that and stir the mixture overnight. Neutralize thereaction mixture with AcOH and remove the solvents under reducedpressure. Purify the residue by silica gel chromatography (DCM, 20%MeOH/DCM) to get the required product 318.

Preparation of 319:

Galactose trichloroacetimidate 315 (10.00 g, 13.53 mmol) and the azidoalcohol 306 (2.60 g, 14.88 mmol) were co evaporated with toluene (2×40ml) two times and dried under high vacuum. Anhydrous ether (50 mL) andmolecular sieves (20 g) were added to the solution and cooled in an icebath. TMSOTf (0.300 g, 1.349 mmol) was added and the mixture was stirredfor 15 minutes, TLC checked and quenched the reaction mixture with TEA.Solvent was removed and the residue purified by silica gel columnchromatography (gradient elution: 20-60% ethylacetate/hexanes) to getthe required compound 319 as a color less liquid (8.623 g, 85%). MS: MWCalc. for C₄₀H₃₉N3O₁₂: 753.25. Found 776.30 (M+Na).

Preparation of 320:

Compound 319 (4.19 g, 5.55 mmol) was dissolved in a mixture ofDCM/MeOH(1:2, 50 mL) and sodium methoxide solution (55 mL, 0.5 M inMeOH) was added to that and stir the mixture overnight. Neutralize thereaction mixture with AcOH and removed the solvents under reducedpressure. Residue was purified by silica gel chromatography (DCM, 20%MeOH/DCM) to get the required product 320 as a color less liquid (1.80g, 96%). MS: MW Calc. for C₁₂H₂₃N₃O₈: 337.15. Found 360.15 (M+Na).

Preparation of 322:

Mannose trichloroacetimidate 321 (15.23 g, 20.55 mmol) and 302 (4.36 g,1.02 eq.) were dissolved in Toluene and aziotroped two times. Theresidue dried under high vacuum overnight. Anhy. diethyl ether (30 mL)and Molecular sieves (10 g) were added to that. Reaction mixture cooledin an ice-water bath. TMSOTf (0.5 mL, 0.1 eq) was added to that andstirred the mixture for 10 minutes. Reaction was monitored by TLC andquenched with TEA. Filtered of the molecular sieves and solvents wereremoved under reduced pressure. Residue was purified by chromatography(hexane, 15-25% EtOAc/Hexane) to get compound 322 as colorless liquid(14.52 g, 90%). MS: Calculated for C₄₆H₄₂O₁₂, 786.27. Found 809.25((M+Na).

Preparation of 323:

Mannose benzyl ester (14.30 g, 18.17 mmol) was dissolved in Ethylacetate (100 mL) to that two drops of acetic acid was added. Degassed,Pd/C (1.50 g, 10 wt % Degussa wet type) was added and hydrogenated underballoon pressure for 24 hrs. Reaction was monitored by TLC and MALDI. Itwas filtered through a small pad of celite, washed with ethyl acetate.Solvent was removed and the residue dried under high vacuum to get thecompound as color less oil (11.20 g, 90%). MS: Calculated for C₃₉H₃₆O₁₂,696.22. Found 719.18 (M+Na). Crude acid (4.40 g, 6.35 mmol) andpropargyl amine (0.384 g, 6.99 mmol) and HBTU (2.40 g, 6.35 mmol) aredissolved in DMF (50 mL). DIEA is added and stir the reaction mixtureovernight. Solvent is removed and the residue is purified by silica gelchromatography to get the required product 323.

Preparation of 324:

Compound 323 (2.00 g, 2.72 mmol) is dissolved in a mixture ofDCM/MeOH(1:2, 50 mL) and sodium methoxide solution (20 mL, 0.5 M inMeOH) is added to that and stir the mixture overnight. Neutralize thereaction mixture with AcOH and remove the solvents under reducedpressure. Purify the residue by silica gel chromatography to get therequired product 324.

Preparation of 325:

Mannose trichloroacetimidate 321 (15.00 g, 20.24 mmol) and azido alcohol306 (4.25 g, 1.2 eq) were dissolved in Toluene and aziotroped twice. Theresidue dried under high vacuum overnight. Anhydrous diethyl ether (30mL) and Molecular sieves (10 g) were added to mixture. Reaction mixturewas cooled in an ice-water bath. TMSOTf (0.5 mL, 0.1 eq) was added andstirred the mixture for 10 minutes. Reaction was monitored by TLC andquenched with TEA. Filtered of the molecular sieves and solvents wereremoved under reduced pressure. Residue was purified by silica gelcolumn chromatography (20-50% EtOAc/Hexane) to obtain compound 325 ascolorless liquid (8.36 g, 55%). ¹H NMR (DMSO-d₆, 400 MHz) δ=8.05 (d,J=7.2 Hz, 2H), 7.19-7.92 (m, 18H), 6.00 (t, J=10.4 Hz, 1H), 5.72 (dd,J=3.2, 10.4 Hz, 1H), 5.62 (dd, J=2, 3.2 Hz, 1H), 5.25 (d, J=1.6 Hz, 1H),4.50-4.65 (m, 3H), 3.60-3.95 (m, 10H), 3.36 (t, J=4.8 Hz, 2H). MS: MWCalc. for C₄₀H₃₉N₃O₁₂: 753.25. Found 776.23 (M+Na).

Preparation of 326:

Compound 319 (5.30 g, 7.03 mmol) was dissolved in a mixture ofDCM/MeOH(1:2, 50 mL) and sodium methoxide solution (70 mL, 0.5 M inMeOH) was added to that and stir the mixture overnight. Neutralize thereaction mixture with AcOH and removed the solvents under reducedpressure. Residue was purified by silica gel chromatography (DCM, 20%MeOH/DCM) to get the required product 320 as a color less liquid (1.98g, 84%). MS: MW Calc. for C₁₂H₂₃N₃O₈: 337.15. Found 360.15 (M+Na)

Preparation of 329:

mPEG₂₀₀₀ (20.00 g, 10.00 mmol) and TEA (4.08 mL, 3 eq) were dissolved inDCM (100 mL) and cooled in an ice bath. To that a solution of tosylchloride (3.00 g, 1.45 eq) in DCM(20 mL) was added drop wise and stirredthe mixture overnight. Diluted with DCM, washed with water and saturatedsodium bicarbonate solution. Dried over sodium sulfate and removed thesolvent in vacuo to get the required tosylate 328. Tosylate and NaN₃ (25g) were taken in DMF and heated at 100° C. in pressure bottle overnight.Transferred to an RB flask and removed the solvent under reducedpressure. Triturated with DCM and filtered through sintered funnelwashed with DCM. Crude product was purified by silica gel chromatographyto get the required product as a white powder (18.10 g, 82%).

Preparation of 330:

mPEG₂₀₀₀ 327 (20.00 g, 10.00 mmol) was dissolved in DMF (100 ml) andcooled in ice bath. NaH(1.00 g, excess, 60%) was added and stirred themixture for 30 minutes. Propargyl bromide (3.00 g, 1.5 eq) and TBAI(2.00 g) were added and stirred mixture overnight. Quenched with icecold water and removed the solvents under reduced pressure. Dissolvedthe residue in DCM and washed with water and dried over sodium sulfate.Crude product was purified by silica gel chromatography to get therequired product 330 (18.20 g, 81%) as pale brown solid.

Preparation of 402:

Z-amino caproic acid 401 (1.00 g, 3.76 mmol) is dissolved in DMF (50mL). To that HBTU (1.43 g, 3.76 mmol) and DIEA (3.26 mL, 5.00 eq.) isadded and stir the mixture for few minutes. Dissolve mannose amine 400(7.41 g, 3.00 mmol) in 50 ml of DMF and add that to the reactionmixture, stir for 48 hrs. Solvents are removed in vacuo and the residuedissolve in DCM, wash with NaHCO₃ solution and water. Dry over anhydroussodium sulfate and the solvents are removed under reduced pressure.Purify the compound by silica gel chromatography to get the requiredcompound 402.

Preparation of 403:

Took compound 402 (3.00 g, 1.10 mmol) in methanol (50 mL), to that 1 mLof acetic acid is added. Hydrogenate under balloon pressure using Pd/C(0.300 g, 10 wt % Degussa wet type) to get the compound 403.

Preparation of 404:

Compound 403 (2.00 g, 0.776 mmol) reacts with propynoic acid underpeptide coupling conditions to get the compound 404.

Preparation of 405:

Compound 404 (1.00 g, 0.380 mmol) is dissolved in MeOH(10 mL) and sodiummethoxide solution (10 mL, 0.5 M in MeOH) is added to that and stir themixture overnight. Reaction is monitored by TLC and the mixtures passthrough a column of CM Sepharose resin. Wash with MeOH and remove thesolvents in vacuo to get the product 405.

Preparation of 406:

Compound 403 (2.00 g, 0.776 mmol) reacts with Triflic anhydride andsodium azide to get the compound 406.

Preparation of 407:

Compound 406 (1.00 g, 0.384 mmol) is dissolved in MeOH(10 mL) and sodiummethoxide solution (10 mL, 0.5 M in MeOH) is added to that and stir themixture overnight. Reaction is monitored by TLC and the mixtures passthrough a column of CM Sepharose resin. Wash with MeOH and remove thesolvents in vacuo to get the product 407

Preparation of 409:

Z-amino caproic acid 401 (2.19 g, 8.25 mmol) was dissolved in DMF (50mL). To that HBTU (3.13 g, 8.25 mmol) and DIEA (7.19 mL, 5.00 eq.) wasadded and stirred the mixture for few minutes. GalNAc amine 408 (10.10g, 5.52 mmol) was dissolved in 50 ml of DMF and was added to reactionmixture, stirred for 48 hrs. TLC and MALDI were checked for productformation. Solvents were removed in vacuo and the residue was dissolvedin DCM, washed with NaHCO₃ solution and water. Dried over anhydroussodium sulfate and the solvents were removed under reduced pressure.Residue was purified by silica gel column chromatography (eluted withethyl acetate, followed by gradient elution of 5-15% MeOH/DCM) to getthe required compound 409 as off white solid (6.20 g, 57%). ¹HNMR(DMSO-d₆ 400 MHz) δ=7.89 (t, J=5.12 Hz, 3H), 7.80 (d, J=9.27 Hz, 3H),7.15-7.40 (m, 7H), 5.20 (d, J=3.41 Hz, 3H), 4.98 (s, 2H), 4.95 (dd,J=3.40, 11.30 Hz, 3H), 4.54 (d, J=8.64 Hz, 3H), 3.72-4.10 (m, 12H),3.10-3.65 (m, 52H), 2.28 (t, J=6.20 Hz, 6H), 2.09 (s, 9H), 1.98 (s, 9H),1.88 (s, 9H), 1.76 (s, 9H), 1.09-1.30 (m, 6H). MS: MW Calc. forC₈₇H₁₃₆N₈O₄₂: 1964.88. Found 1987.75 (M+Na).

Preparation of 410:

Compound 409 (6.10 g, 3.10 mmol) was dissolved in methanol (50 mL), tothat 1 mL of acetic acid was added. Degassed the reaction mixture, Pd/C(0.700 g, 10 wt % Degussa wet type) was added to the solution andhydrogenated under balloon pressure for 36 hrs. Reaction mixture wasfiltered through a small pad of celite, washed with MeOH. To thefiltrate, 1.25 eq of TFA and toluene (50 mL) were added and solventsremoved under reduced pressure. The residue was co-evaporated withtoluene two times and dried under high vacuum overnight night to get therequired compound 410 as an off white solid (6.10 g, quantitative). Thiscompound used as such for the next reaction with out any furtherpurification. MS: MW Calc. for C₇₉H₁₃₀N₈O₄₀: 1830.84. Found 1853.81(M+Na).

Preparation of 411:

Compound 410 (2.00 g, 1.09 mmol) reacts with Tf₂O and sodium azide toget compound 411.

Preparation of 412:

Compound 411 (1.00 g, 0.539 mmol) is dissolved in MeOH(10 mL) and sodiummethoxide solution (10 mL, 0.5 M in MeOH) is added to that and stir themixture overnight. Reaction is monitored by TLC and the mixtures passthrough a column of CM Sepharose resin. Wash with MeOH and remove thesolvents in vacuo to get the product 412.

Preparation of 414:

Compound 413 (3.30 g, 5.65 mmol) was dissolved in a mixture ofMeOH/EtOAc (60 mL, 1:2); Pd/C (500 mg, Degussa wet type) was added andhydrogenated under balloon pressure over night. Filtered the reactionmixture through a small pad of celite and removed the solvents to getthe required product 414. The crude product used for the next reactionwith out further purification. MS: MW Calc. for C₁₆H₃₃N₃O₃: 315.25.Found 316.22 (M+H)

Preparation of 416:

GalNAc acid 415 (5.81 g, 12.99 mmol) and HBTU (4.976 g, 13.02 mmol) weredissolved in DMF (50 mL) to that DIEA (6.79 mL, 5 eq) was added andstirred the mixture for few minutes. To that a solution of compound 414in DMF was added and stirred overnight at room temperature. Solventswere removed in vacuo and the residue was dissolved in DCM, washed withNaHCO₃ solution and water. Dried over anhydrous sodium sulfate and thesolvents were removed under reduced pressure. Residue was purified bysilica gel column chromatography (eluted with ethyl acetate, followed bygradient elution of 3-10% MeOH/DCM) to get the required compound 416 asoff white solid (5.25 g, 79%). MS: MW Calc. for C₅₄H₈₇N₅O₂₃: 1173.58.Found 1196.60 (M+Na).

Preparation of 417:

Compound 416 (5.15 g, 4.40 mmol) was dissolved in DCM (30 mL) to thatTFA(30 ml in 20 mL of DCM) was added and stirred for 2 hr's at roomtemperature. TLC checked and removed the solvents. Co-evaporated withtoluene two times and dried under high vacuum. Crude product used forthe next reaction. MS: MW Calc. for C₅₀H₇₉N₅O₂₃: 1117.52. Found 1140.55(M+Na).

Preparation of 419:

Compound 419 is prepared from 418 and 417 using HBTU mediated peptidecoupling.

Preparation of 420:

Compound 419 (1.00 g, 0.805 mmol) is dissolved in MeOH(10 mL) and sodiummethoxide solution (10 mL, 0.5 M in MeOH) is added to that and stir themixture overnight. Reaction is monitored by TLC and the mixtures passthrough a column of CM Sepharose resin. Wash with MeOH and remove thesolvents in vacuo to get the product 420.

Preparation of 422:

Compound 422 is prepared from 421 and 417 using HBTU mediated peptidecoupling.

Preparation of 423:

Compound 422 (1.00 g, 0.866 mmol) is dissolved in MeOH(10 mL) and sodiummethoxide solution (10 mL, 0.5 M in MeOH) is added to that and stir themixture overnight. Reaction is monitored by TLC and the mixtures passthrough a column of CM Sepharose resin. Wash with MeOH and remove thesolvents in vacuo to get the product 423.

Example 8 Folate Building Blocks for Click-Chemistry

In order to synthesize azido functional group containing folateconjugates the following strategy was used. The azido amine tether 334was synthesized starting from the commercially available diamine 331 asshown in Scheme 10.

Synthesis of Amine 332:

A solution of (Boc)₂O (66 g, 0.303 mol) in dioxane (1 L) was added in adropwise fashion over 4 h to a cooled (0° C.) solution of the diamine(400 g, 1.82 mol) in dioxane (1 L), and the mixture was stirred at roomtemperature overnight. Aqueous workup then column chromatographyprovided the pure mono Boc amine 332 (83 g, 86%). MS: MW Calc. forC₁₅H₃₂N₂O₅: 320.42. Found 321.41 (MH⁺).

Synthesis of Azide 333:

The triflic azide stock solution was prepared as reported in TetrahedronLetters 47 (2006) 2382-2385. The amine (0.96 g, 3 mmol), sodiumbicarbonate (0.85 mg, 10 mmol) and copper (II) sulfate pentahydrate (22mg, 0.1 mmol) were dissolved in water (3 mL). Triflic azide stocksolution (5 mL) was added, followed by the addition of methanol (20 mL)to yield a homogeneous system. The blue mixture was stirred for 30 minafter which the TLC and MS showed the complete disappearance of startingamine. The reaction mixture was concentrated in a rotary evaporator andthe residue was purified by chromatography on silica gel (eluent:dichloromethane-methanol) to obtain the pure azide 333 (1 g, 96%) as anoil. MS: MW Calc. for C₁₅H₃₀N₄O₅: 346.42. Found 347.41 (MH⁺). ¹HNMR(CDCl₃, 400 MHz) δ=4.68 (bs, 1H), 3.40-3.30 (m, 12H), 3.16 (t, J=6.4 Hz,2H), 3.00-2.95 (m, 2H), 1.68-1.54 (m, 4H), 1.04 (s, 9H).

Synthesis of 334:

The azide 333 (1 g, 2.88 mmol) was dissolved in ethanol (10 mL) and tothis a 2M solution of HCl in ether was added and the mixture was stirredat room temperature overnight. The MS showed the absence of startingmaterial. The reaction mixture was concentrated and the thus obtainedoil was used as such for the next reaction without further purification.MS: MW Calc. for C₁₀H₂₃C1N₄O₃: 246.17. Found 247.17 (MH⁺). ¹HNMR(DMSO-d₆ 400 MHz) δ=8.96 (bs, 1H), 7.92 (bs, 2H), 3.52-3.40 (m, 12H),3.37 (t, J=6.8 Hz, 2H), 2.85-2.77 (m, 2H), 1.81-1.70 (m, 4H).

The commercially available pteroic acid 335 was transiently protectedwith t-butyldiphenyl silyl group and treated with isobutyric anhydridefollowed by acidic workup provided the exocyclic amine protected pteroicacid 336 which on coupling with methyl t-butyl ester of glutamic acidprovided the fully protected folic acid 338. Hydrolysis of the t-butylester with TFA provided the acid 339 which was coupled with the azidoamine tether 334 to give the couple product 340 as an oil. Treatment ofthis compound with lithium hydroxide followed by methylamine providedthe azido compound 341.

Synthesis of 336:

To a suspension of pteroic acid (25 g, 61.2 mmol) and DMAP (11.25 g, 92mmol) in anhydrous pyridine (400 mL), TBDPS chloride (42 g, 153 mmol)was added. The reaction mixture was stirred at room temperature for 30 hafter which isobutric anhydride (14.6 g, 92 mmol) was added and themixture was slightly warmed. An additional 60 mL of pyridine was alsoadded and the reaction mixture was stirred at room temperatureovernight. The reaction mixture became homogenous after which pyridineand other volatiles were concentrated in a rotary evaporator. Theresidue was stirred with EtOAc (1 L) and acetic acid (100 mL) and water(500 mL) for 24 h. The thus obtained slurry was filtered, the residuewas washed with water (500 mL), EtOAc (1 L) and dried to obtain the pureproduct as a white solid (26.1 g, 89%). ¹H NMR (DMSO-d₆, 400 MHz) δ=8.87(s, 1H), 7.95 (d, J=8.6 Hz, 2H), 7.67 (d, J=8.6 Hz, 2H), 5.21 (s, 2H),2.79-2.74 (m, 1H), 1.12 (d, J=6.83 Hz, 6H), ¹³C NMR (DMSO-d₆) δ=180.72,166.49, 159.25, 149.87, 147.68, 142.69, 136.34, 134.45, 130.54, 129.16,128.86, 127.49, 34.96, 33.09, 26.52, 18.88, 18.74. ¹⁹F NMR (DMSO-d₆)δ-64.32. MS. Molecular weight calculated for C₂₀H₁₇F₃N₆O₅, Cal. 478.12.Found 479.12 (MH⁺).

Synthesis of 338:

In a representative procedure, the pteroic acid precursor 336 (2.4 g, 5mmol) was dissolved in anhydrous DMF (20 mL), HBTU (1.9 g, 1 eq.)followed by DIEA (1 mL, 5 eq.) were added and stirred for 20 minutes. Tothis reaction mixture the amine hydrochloride 337 (1.2 g, 1 eq) wasadded as a solution in DMF (6 mL). Reaction was monitored by TLC (8%MeOH/DCM, PMA stain). TLC of the reaction mixture showed completion ofthe reaction. The reaction mixture was slowly poured in ice withvigorous stirring. The precipitated product was filtered to get theproduct 338 as a white solid (Yield=2.85 g, 86%). ¹H NMR (DMSO-d₆, 400MHz) δ=12.33 (s, 1H), 11.94 (s, 1H), 8.88 (s, 1H), 8.82 (d, J=7.3 Hz,1H), 7.90 (d, J=8.6 Hz, 2H), 7.68 (d, J=8.4 Hz, 2H), 5.22 (s, 2H),4.46-4.40 (m, 1H), 3.62 (s, 3H), 2.86-2.73 (m, 1H), 2.32 (t, J=7.4 Hz,2H) 2.05-1.90 (m, 2H), 1.35 (m, 9H), 1.12 (d, J=6.8 Hz, 6H). ¹³C NMRDMSO-d₆) δ=180.75, 172.13, 171.45, 165.64, 159.10, 154.80, 149.97,149.79, 147.72, 141.75, 134.15, 130.53, 128.70, 128.49, 117.50, 114.64,79.79, 51.96, 51.91, 34.96, 31.22, 27.68, 25.71, 18.72. MS. Molecularweight calculated for C₃₀H₃₄F₃N₇O₈, Cal. 677.63. Found 676.72 (M−H⁻).

Synthesis of 339:

The ester 338 (2 g, 2.9 mmol) was dissolved in 20 mL of 50% TFA indichloromethane and the solution was stirred at room temperature for 30min. after which the TLC showed the complete disappearance of thestarting ester. The reaction mixture was concentrated and the residuewas crystallized from CH₂Cl₂:Hexanes (2:3) and crystallized product wasfiltered off and dried to obtain the pure product 339 (1.76 g, 96%) asoff white powder. ¹H NMR (DMSO-d₆, 400 MHz) δ=12.32 (bs, 1H), 11.94 (s,1H), 8.88 (s, 1H), 8.84 (d, J=7.4 Hz, 1H), 7.90 (d, J=8.3 Hz, 2H), 7.69(d, J=8.3 Hz, 2H), 5.22 (s, 2H), 4.45-4.41 (m, 1H), 3.62 (s, 3H),2.78-2.75 (m, 1H), 2.35 (t, J=7.4 Hz, 2H) 2.07-1.92 (m, 2H), 1.12 (d,J=6.8 Hz, 6H). ¹³C NMR DMSO-d₆) δ=180.77, 173.70, 172.19, 165.70,159.21, 155.54, 149.93, 149.84, 147.75, 141.78, 134.18, 130.53, 128.71,128.49, 117.51, 114.64, 53.98, 52.06, 51.93, 34.97, 30.11, 25.68, 18.73.MS. Molecular weight calculated for C₂₆H₂₆F₃N₇O₈, Cal. 621.18. Found620.18 (M−H⁻).

Synthesis of 340:

Coupling of the amine 334 (0.6 g) with the acid 339 (1.2 g) using asimilar procedure to that used for the synthesis of 338 provided thecoupled azide 340 (1.68 g, 93%) as a light yellow foam. ¹H NMR (DMSO-d₆,400 MHz) δ=12.34 (s, 1H), 11.95 (s, 1H), 8.89 (s, 2H), 7.92 (d, J=8.4Hz, 2H), 7.81 (m, 1H), 7.70 (d, J=8.4 Hz, 2H), 5.22 (s, 2H), 4.40-4.34(m, 1H), 3.62 (s, 3H), 3.50-3.31 (m, 15H), 3.09-3.00 (m, 2H), 2.80-2.72(m, 1H), 2.20 (t, J=7.4 Hz, 2H) 2.10-1.89 (m, 2H), 1.76-1.54 (m, 4H),1.12 (d, J=6.8 Hz, 6H). MS. Molecular weight calculated forC₃₆H₄₆F₃N₁₁O₁₀, Cal. 849.81. Found 850.2 (MH⁺).

Synthesis of 341:

The azide 340 (1 g) was dissolved in THF (20 mL) and to it an aqueoussolution of lithium hydroxide (100 mg in 2 mL of water) was added andthe solution was stirred at room temperature for 4 h after which the MSshowed the complete disappearance of SM. The reaction mixture wasacidified to pH 5 using acetic acid and the RM was diluted with ethylacetate (100 mL). The precipitated product was filtered off and washedwith water and ethyl acetate and dried under vacuo at 40° C. overnightto get the pure azide 341 (0.455 g 55%) as an orange solid. ¹H NMR(DMSO-d₆, 400 MHz) δ=8.59 (s, 1H), 7.85 (bs, 1H), 7.72 (bs, 1H), 7.56(d, J=8.4 Hz, 2H), 6.88 (bs, 1H), 6.65 (d, J=8.4 Hz, 2H), 4.45 (s, 2H),4.00-4.02 (m, 1H), 3.50-3.33 (m, 14H), 3.04-3.00 (m, 2H), 2.07-1.83 (m,4H), 1.76-1.54 (m, 4H). MS. Molecular weight calculated for C₂₉H₃₉N₁₁O₈,Cal. 669.69. Found 668.2 (M−H⁻).

In another embodiment, we have synthesized the alkyne containing folicacid is synthesized as follows. In this case the protected pteroic acid335 was coupled with the protected lysine 342 to get the coupled product343 which on Cbz deprotection provided the amine 344. Coupling of theamine 344 with the acid 350 provided the coupled product 345 which afterpurification and deprotection provided the product 346 as describedbelow.

Synthesis of 343:

Using a similar procedure to that used for the synthesis of 338,coupling of the acid 335 with the lysine derivative 342 provided thecoupling product 343 as a white solid in 95% yield.

Synthesis of 344:

The compound 343 on hydrogenation with Pd/C provided the deprotectedamine 344 as a yellow solid.

Synthesis of 345:

Coupling of the amine 344 with the acid 350 using a procedure to thatused for the synthesis of 338 provided the couple product 345 in highyields.

Synthesis of 346:

The deprotection of the protecting groups is achieved using a similarprocedure as described for the synthesis of 341 to isolate the fullydeprotected alkyne 346.

In another embodiment the folate conjugated oligo building block issynthesized as follows. The synthesis of the building block 362 iscarried out using a similar procedure to that of 115 and the synthesisof the amidite is carried out as described for the synthesis of 121.

Example 9 Lipid Building Blocks for Click Chemistry

The following acetylinic CPG supports and phosphoramidites were preparedin the usual way.

Preparation of Compound 352

A solution of compound 351 (8 g, 15 mmol) in DCM (300 mL) was treatedsuccessively with Hünig's base (2.5 eq), 350 (1.1 eq) and HBTU (1.05eq). The solution was stirred at r.t. overnight. Aqueous workup thencolumn chromatography gave pure compound 352. Yield 7.3 g, 78%.

Preparation of Compound 357

Compound 357 was prepared using a procedure analogous to that describedfor compound 352.

Preparation of Compound 354

Compound 354 was prepared using a procedure analogous to that describedfor compound 358. Yield 5.7 g, 84%.

Preparation of Compound 358

A solution of Hünig's base (2 eq) and compound 357 (5.0 g, 9.7 mmol) inDCM (50 mL) was treated with amidite reagent (1.5 eq). After stirringfor 10 min, TLC showed complete reaction. Aqueous workup then columnchromatography gave pure compound 358 (6.1 g, 88%).

Preparation of Compound 359

A solution of compound 357 (2.07 g, 4.03 mmol) in DCM (150 mL) wastreated with DMAP (3 eq) then succinic anhydride (2 eq) and stirred atr.t. overnight. Column chromatography gave the intermediate succinate(2.5 g, 87%) as a ⁺HNEt₃ salt. A solution of this intermediate (2.49 g,4.07 mmol) in DMF (200 mL) was treated successively with Hünig's base (5eq), HBTU (1 eq) then 500 Å, 140 μmol/g CPG-NH₂ (29.1 g, 1 eq). Aftershaking for 1 h, the support was collected by filtration then subjectedto capping with Ac₂O/py (4:1, 200 mL) by shaking for 1 h. Aftercollection by filtration and drying in vacuo, 22.4 g of 359 wasobtained. Loading calculated to be 90 μmol/g.

Preparation of Compound 355

Compound 354 was prepared using a procedure analogous to that describedfor compound 359. Obtained 22.4 g with loading 91 μmol/g.

Preparation of Compound 361

Compound 361 was prepared using a procedure analogous to that describedfor compound 352.

Preparation of compound 368

Spermine 366 (6.0 g, 30 mmol) was dissolved in MeOH (350 mL), cooled at-78° C. and ethyltrifluoro acetate (4.1 mL, 35 mmol) was added. Thesolution was warmed to r.t. and stirred for 1 h whereon a solution ofBoc₂O (30.5 g, 410 mmol) in MeOH (30 mL) was added dropwise to the crudeintermediate 367. After 16 h at r.t., the mixture was hydrolyzed byaddition of aqueous NaOH. Extraction into EA then column chromatographygave pure compound 3 (5.7 g, 38%).

Example 10 Glycolipid Building Blocks for Click Chemistry

Acetylene unit was coupled with the lithocholic derivative 500 via apeptide linkage to give 501. After hydrolysis, GalNAc unit will becoupled with 502 to give 503.

Compound 501: To a solution of 5-hexynoic acid (907 mg, 8.09 mmol) inDMF (50 mL), HBTU (3.07 g, 8.09 mmol) and iPr₂NEt (6.71 mL, 38.5 mmol)were added. After 5 minute, compound 500 (3.0 g, 7.7 mmol) was added tothe solution. The reaction mixture was stirred for 14 hours at roomtemperature. After extraction with Et₂O (300 mL) and NaHCO₃ aq. (150mL), purification by silica gel column chromatography (Hexane:EtOAc=2:1,R_(f)=0.37) gave compound 501 (3.63 g, 7.50 mmol, 98%). ¹H NMR (DMSO-d₆,400 MHz) δ 7.70 (d, J=7.2 Hz, 1H), 3.95-3.97 (m, 1H), 3.57 (s, 3 H),2.78 (t, J=2.6 Hz, 1H), 0.86-2.34 (m, 40H), 0.61 (s, 3H). ¹³C NMR(DMSO-d₆, 100 MHz) δ□ 173.6, 170.7, 84.1, 71.2, 55.9, 55.4, 51.1, 44.2,42.2, 39.1, 36.3, 35.1, 34.7, 34.5, 34.1, 30.5, 30.3, 27.6, 26.3, 25.7,24.5, 24.4, 23.7, 23.4, 20.6, 18.0, 17.4. Molecular weight for C₃₁H₅₀NO₃(M+H)⁺ Calc. 484.38. Found 484.3.

Commercially available azide unit 504 was coupled with the lithocholicderivative 500 to give 505. After hydrolysis, 506 will be coupled withthe GalNAc unit to give a glycolipid molecule containing azide group507.

Oleyl-lithocholic acetylene derivative 509 will be prepared from 508 bystandard peptide coupling using propargylamine. Commercially availablediamine will be protected and converted to the corresponding azidederivative 512. The azide will be coupled with 508 to generateoleyl-lithocholic azide derivative 513.

Another oleyl-lithocholic derivative 514 will be functionalized withpropargylamine and azide compound 512 to give 515 and 516, respectively.

The lithocholic ester 517 was coupled with propargylamine to afforcompound 518. Conjugation of 517 with azide compound 512 via a carbamatelinkage to give 519.

Compound 518: The activation of the hydroxyl group of compound 517 wascarried out using N,N′-disuccinimidyl carbonate and triethylamine inCH₂Cl₂. After aqueous work-up, the crude material (3.03 g, 5.88 mmol)was dissolved in CH₂Cl₂ (60 mL). Then, triethylamine (1.64 mL, 11.76mmol) and propargylamine (0.524 mL, 7.64 mmol) were added to thesolution. The reaction mixture was stirred for 14 hours at roomtemperature. Aqueous work-up and purification by column chromatography(Hexane:EtOAc=5:1 to 2:1) gave compound 518 (2.25 g, 4.77 mmol, 81%). ¹HNMR (DMSO-d₆, 400 MHz) δ 7.44 (t, J=5.5 Hz, 1H), 4.43-4.51 (m, 1H),3.73-3.75 (m, 2H), 3.57 (s, 3H), 3.08 (t, J=2.2 Hz, 1H), 2.16-2.36 (m,2H), 0.86-1.93 (m, 32H), 0.61 (s, 3H). ¹³C NMR (DMSO-d₆, 100 MHz) δ□173.6, 155.5, 81.5, 73.6, 72.7, 55.9, 55.4, 51.1, 42.2, 41.2, 39.9,35.3, 34.7, 34.5, 34.1, 32.3, 30.5, 30.3, 29.6, 27.6, 26.6, 26.5, 25.9,23.7, 23.0, 20.3, 18.0, 11.8. Molecular weight for C₂₉H₄₅NNaO₄ (M+Na)⁺Calc. 494.32. Found 494.2.Oleyl-lithocholic GalNAc building block will be synthesized according toScheme 19. Compound 523 was prepared from compound 508 (Rensen, P. C. N.et al., J. Med. Chem., 2004, 47, 5798-5808). Briefly, standard peptidecoupling of 508 with ethyl iminodiacetate gave 520. The ester hydrolysisgenerated the corresponding di-acid 521. The di-acid was coupled withGalNAc via anhydro intermediate 522 to give 523.523 will befunctionalized with propargylamine and azide compound 512 to give 524and 525, respectively.

Preparation of Compound 520:

A solution of compound 508 (10.5 g, 16.4 mmol, Rensen, P. C. N. et al.,J. Med. Chem., 2004, 47, 5798-5808) and HBTU (6.54 g, 17.2 mmol) inCH₂Cl₂ (170 mL) was treated successively with iPr₂NEt (14.3 mL, 82.1mmol) and diethyl iminodiacetate (3.26 g, 17.2 mmol) then allowed tostir for 69 hours at room temperature. Aqueous work-up then silica gelcolumn chromatography (hexane:ethyl acetate=1:1, R_(f)=0.47) gavecompound 520.

Yield: 11.1 g, 83%. ¹H NMR (CDCl₃, 400 MHz) δ 5.66 (d, J=8.0 Hz, 1H),5.33-5.36 (m, 2H), 4.14-4.26 (m, 9H), 0.86-2.38 (m, 71H), 0.64 (s, 3H).¹³C NMR (CDCl₃, 100 MHz) δ 174.0, 172.1, 169.4, 169.0, 129.9, 129.7,61.6, 61.2, 60.8, 56.4, 56.1, 50.2, 47.9, 44.9, 42.7, 40.1, 39.7, 38.1,37.1, 35.6, 35.3, 35.0, 31.9, 31.4, 30.9, 30.6, 29.7, 29.67, 29.60,29.5, 29.3, 29.24, 29.22, 29.1, 28.1, 27.2, 27.1, 26.7, 26.1, 25.9,24.8, 24.1, 22.6, 21.0, 18.4, 14.18, 14.16, 14.11, 14.09, 12.0.Molecular weight for C₅₀H₈₇N₂O₆ (M+H)⁺ Calc. 811.66. Found 811.6.

Preparation of Compound 521:

Compound 520 (6.05 g, 7.46 mmol) was treated with lithium hydroxidemonohydrate (1.25 g, 29.8 mmol) in THF (110 mL) and H₂O (22 mL). After 4hours, 70 mL of Amberlite IR-120 (plus) ion exchange resin was addedthen stirred for 10 minutes. The resulting clear solution was filtered,washed with THF/H₂O and evaporated. Co-evaporation with toluene gave thecompound 521 as a white solid.

Yield: 4.81 g, 85%. ¹H NMR (CDCl₃, 400 MHz) δ 5.99 (d, J=7.2 Hz, 1H),5.32-5.36 (m, 2H), 4.13-4.24 (m, 5H), 0.86-2.37 (m, 65H), 0.64 (s, 3H).¹³C NMR (CDCl₃, 100 MHz) δ 175.3, 173.7, 173.0, 171.5, 130.0, 129.7,107.9, 67.7, 56.4, 55.9, 45.6, 42.7, 40.1, 39.8, 38.1, 36.9, 35.6, 35.3,35.0, 31.9, 30.9, 30.4, 29.8, 29.75, 29.70, 29.6, 29.5, 29.4, 29.3,29.2, 29.1, 28.2, 27.3, 27.2, 26.7, 26.1, 26.0, 25.9, 24.2, 24.1, 23.9,22.7, 21.0, 18.4, 14.1, 12.0. Molecular weight for C₄₆H₇₇N₂O₆ (M−H)⁻Calc. 753.58. Found 753.5.

Preparation of Compound 523:

To a solution of compound 521 (583 mg, 0.772 mmol) in CH₂Cl₂ (25 mL), 1M DCC solution of CH₂Cl₂ (0.772 mL, 0.772 mmol) was added at roomtemperature and the reaction mixture was stirred for 1.5 hours to formthe intermediate 522. Then, a solution of compound 5 (1.50 g, 0.772mmol) and Et₃N (0.377 mL, 2.70 mmol) in CH₂Cl₂ (5 mL) was added. Thereaction mixture was stirred overnight. The crude was purified by silicagel column chromatography (5% MeOH in CH₂Cl₂ with 2% Et₃N, R_(f)=0.24)to yield the compound 523. Yield: 1.62 g, 79% as the triethylammoniumsalt. ¹H NMR (CDCl₃, 400 MHz) δ 5.68 (d, J=7.2 Hz, 1H), 5.29-5.34 (m,5H), 5.15-5.18 (m, 3H), 4.77-4.79 (m, 3H), 3.41-4.16 (m, 59H), 2.93-3.24(m, 6H), 0.85-2.56 (m, 117H), 0.61 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz) δ174.9, 173.5, 172.2, 171.6, 170.8, 170.43, 170.36, 129.9, 129.7, 101.63,101.58, 70.63, 70.60, 70.5, 70.3, 69.8, 69.69.29, 69.26, 69.22, 69.1,68.75, 68.67, 67.3, 66.80, 66.75, 62.9, 61.6, 59.6, 56.43, 56.37, 56.1,56.0, 53.4, 52.7, 50.63, 50.56, 45.6, 44.9, 42.7, 40.11, 40.07, 40.05,39.8, 39.7, 39.30, 39.27, 39.1, 38.1, 37.1, 36.5, 35.6, 35.44, 35.38,35.0, 31.9, 31.4, 30.6, 29.72, 29.67, 29.5, 29.3, 29.2, 29.1, 28.2,27.2, 27.1, 26.7, 26.1, 25.9, 25.3, 24.8, 24.15, 24.11, 23.2, 22.6,21.0, 20.9, 20.7, 18.5, 14.1, 12.0, 8.5, 7.7. MALDI-TOF MS calcd forC₁₂₅H₂₀₅N₁₀O₄₅Na (M+Na)⁺ 2589.40. Found 2588.76.

Pseudouridine Building Blocks for Click Chemistry

N1 at pseudouridine was alkylated with methyl acrylate to give 526. Thisester was hydrolyzed to give the corresponding acid derivative 527. Astandard peptide coupling of 527 with propargylamine and azide compoundwill give 528 and 529 for conjugation via click chemistry, respectively.

Compound 526:

To a solution of pseudouridine (20 g, 81.9 mmol) in 1Mtriethylammoniumbicarbonate buffer (pH 8.5, 780 mL) and EtOH (940 mL),methyl acrylate was dropwisely added. The reaction mixture was stirredovernight. After 16 hours, TLC showed a complete reaction. The solventwas removed and dried in vacuo to give a white foam. The crude materialwas purified by silica gel column chromatography (10% MeOH in CH₂Cl₂,R_(f)=0.23) to give 526 (26.6 g, 80.5 mmol, 98%). ¹H NMR (MeOH-d₄, 400MHz) δ 7.77 (d, J=0.8 Hz, 1H), 4.58 (d, J=4.8 Hz, 1H), 4.15 (t, J=5.2Hz, 1H), 4.05 (t, J=5.0 Hz, 1H), 3.98-4.02 (m, 2H), 3.91-3.94 (m, 1H),3.80 (dd, J=12.0 Hz, 3.3 Hz, 1H), 3.67 (s, 3H), 3.66 (dd, J=12.0 Hz, 3.3Hz, 1H), 2.73-2.77 (m, 2H). ¹³C NMR (CDCl₃, 100 MHz) δ 173.1, 165.4,152.5, 145.8, 112.9, 85.6, 81.5, 75.6, 72.6, 63.3, 52.5, 46.2, 33.7.Molecular weight for C₁₃H₁₉N₂O₈ (M+H)⁺ Calc. 330.11. Found 331.0.

Compound 527:

To a solution of compound 526 (5.00 g, 15.1 mmol) in THF (100 mL) andH₂O (20 mL), lithium hydroxide monohydrate (1.03 g, 25.5 mmol) wasadded. The reaction mixture was stirred overnight. Additional lithiumhydroxide monohydrate (500 mg, 11.9 mmol) was added. After 2 hours, thereaction mixture was treated with Amberlite IR-120 (plus) ion exchangeresin. The resin was filtered off and washed with THF/H₂O. The filtratewas evaporated to give compound 527 as a white solid (4.78 g,quantitatively). ¹H NMR (DMSO-d₆, 400 MHz) δ 11.34 (s, 1H), 7.75 (s,1H), 4.92-4.93 (m, 1H), 4.70-4.72 (m, 1H), 4.45 (d, J=4.0 Hz, 1H),3.80-3.93 (m, 4H), 3.68-3.72 (m, 1H), 3.61 (dd, J=12.0 Hz, 3.2 Hz, 1H),3.47 (dd, J=12.0 Hz, 4.0 Hz, 1H), 3.17 (d, J=3.2 Hz, 1H), 2.59 (t, J=7.0Hz, 2H). ¹³C NMR (DMSO-d₆, 100 MHz) δ 172.1, 163.0, 150.4, 143.6, 111.4,83.2, 79.0, 73.7, 70.4, 61.3, 44.1, 32.7. Molecular weight forC₁₂H₁₅N₂O₈ (M−H)⁻ Calc. 315.08. Found 315.1.

Example 11 Synthesis of Spermine Azide for Conjugation toOligonucleotides

For bioconjugation using click chemistry, azide-containing sperminederivatives were synthesized.

Compound 531:

A suspension of sodium azide (4.36 g, 67 mmol) in acetonitrile (80 mL)was cooled in an ice bath. Then, trifluoromethanesulfonic anhydride(15.7 g, 9.4 mL, 55.6 mmol) was dropwisely added to the mixture using adropping funnel over 10 minute. After the reaction was run for 2 hoursin an ice bath, the reaction mixture was filtered to remove theprecipitation. The precipitation was washed with 20 mL of acetonitrile.The combined acetonitrile solution containing triflic azide was directlyused for the subsequent azidotransfer reaction.

Spermine (530, 11.25 g, 55.6 mmol) was dissolved in acetonitrile (50mL). Then, copper sulfate pentahydrate (139 mg, 0.556 mmol) andtriethylamine (15.5 mL, 111.2 mmol) were added to the solution whilestirring. The mixture was cooled in an ice bath, then, the triflic azidesolution prepared above was added to the mixture slowly. The reactionmixture was allowed to warm to room temperature and run overnight.Di-tert-butyl dicarbonate (60.7 g, 278 mmol) was slowly added to thesolution and the reaction mixture was stirred for 2 hours. Afterevaporation, the crude was extracted with EtOAc and H₂O, dried overanhydrous Na₂SO₄, and purified by silica gel column chromatography(Hexane:EtOAc=2:1, R_(f)=0.46) to give 531 (13.12 g, 24.8 mmol, 45%).

¹H NMR (DMSO-d₆, 400 MHz) δ 6.77 (brs, 1H), 3.30-3.34 (m, 2H), 3.11-3.19(m, 8H), 2.86-2.90 (m, 2H), 1.71-1.72 (m, 2H), 1.54-1.56 (m, 2H),1.37-1.39 (m, 29H). Molecular weight for C₂₅H₄₈N₆NaO₆ (M+Na)⁺ Calc.551.35. Found 551.2.

Compound 531b:

To a solution of compound 531 (1.5 g, 2.84 mmol) in THF (20 mL) and H₂O(1 mL), triphenylphosphine (1.49 g, 5.68 mmol) was added. The reactionmixture was stirred at room temperature overnight. After evaporation,the crude was purified by silica gel column chromatography(CH₂Cl₂:MeOH=4:1, R_(f)=0.70) to give 531b (1.13 g, 2.25 mmol, 79%). ¹HNMR (DMSO-d₆, 400 MHz) δ 6.78 (brs, 1H), 3.10-3.15 (m, 8H), 2.87-2.89(m, 2H), 2.48-2.51 (m, 2H), 1.37-1.55 (m, 35H). Molecular weight forC₂₅H₅₁N₄O₆ (M+H)⁺ Calc. 503.38. Found 503.2.

Compound 532:

To a solution of compound 531 (1.37 g, 2.59 mmol) in CH₂Cl₂ (36 mL),trifluoroacetic acid (4 mL) was slowly added at 0° C. The reactionmixture was stirred for 5 hours at room temperature. After removing thesolvent, the crude was precipitated in Et₂O to give compound 532 (950mg, 1.67 mmol, 64%) as the TFA salt. ¹H NMR (DMSO-d₆, 400 MHz) δ 8.78(brs, 2H), 8.68 (brs, 2H), 7.93 (brs, 3H), 3.48 (t, J=6.4 Hz, 2H),2.94-2.95 (m, 10H), 1.82-1.93 (m, 4H), 1.62 (brs, 4H). Molecular weightfor C₁₀H₂₅N₆ (M+H)⁺ Calc. 229.21. Found 229.3.

Compound 533:

To a solution of compound 532 (324 mg, 0.568 mmol) in pyridine (5 mL),trifluoroacetic anhydride (0.316 mL, 2.27 mmol) was added dropwise at 0°C. The reaction mixture was stirred for 14 hours at room temperature.After extraction with CH₂Cl₂ and NaHCO₃ aq., the crude was purified bysilica gel column chromatography (Hexane:EtOAc=1:1, R_(f)=0.34) to givecompound 533 (214 mg, 0.414 mmol, 73%). ¹H NMR (DMSO-d₆, 400 MHz) δ9.48-9.53 (m, 1H), 3.18-3.43 (m, 12H), 1.75-1.80 (m, 4H), 1.51-1.54 (m,4H). ¹⁹F NMR (376 MHz, DMSO-d₆) δ −70.91, −70.95, −70.97, −71.01,−71.04, −71.15, −71.17, −77.23, −77.25, −77.38. Molecular weight forC₁₆H₂₁F₉N₆NaO₃ (M+Na)⁺ Calc. 539.14. Found 539.0.The extended spermine analogs containing azide group were alsosynthesized.

Compound 535:

A suspension of sodium azide (436 mg, 6.70 mmol) in acetonitrile (8 mL)was cooled in an ice bath. Then, trifluoromethanesulfonic anhydride(0.94 mL, 5.56 mmol) was slowly added to the mixture. After the reactionwas run for 2 hours in an ice bath, the reaction mixture was filtered toremove the precipitation. The precipitation was washed with 20 mL ofacetonitrile. The combined acetonitrile solution containing triflicazide was directly used for the subsequent diazotransfer reaction.

Compound 534 (J. Med. Chem. 2005, 48, 2589-2599; 5 g, 6.97 mmol) wasdissolved in acetonitrile (30 mL). Then, copper sulfate pentahydrate (28mg, 0.112 mmol) and triethylamine (7.75 mL, 55.6 mmol) were added to thesolution. The mixture was cooled in an ice bath, then, the triflic azidesolution prepared above was added to the mixture slowly. The reactionmixture was allowed to warm to room temperature and run overnight. Aftertransferred half of this solution (-32 mL) into a different flask,di-tert-butyl dicarbonate (1.52 g, 6.98 mmol) was slowly added and thereaction mixture was stirred for 2 hours. After evaporation, the residuewas extracted with EtOAc and H₂O, dried over anhydrous Na₂SO₄, filtered,and concentrated in vacuo. The crude was purified by silica gel columnchromatography (Hexane:EtOAc=1:1, R_(f)=0.35) to give 535 (281 mg, 0.333mmol, 5%). ¹H NMR (DMSO-d₆, 400 MHz) δ 6.73 (brs, 1H), 3.05-3.33 (m,18H), 2.86-2.88 (m, 2H), 1.36-1.70 (m, 57H). Molecular weight forC₄₁H₇₈N₈NaO₁₀ (M+Na)⁺ Calc. 865.57. Found 865.5.

Compound 536:

To a solution of compound 535 (275 mg, 0.326 mmol) in CH₂Cl₂ (8 mL),trifluoroacetic acid (2 mL) was added dropwise at 0° C. The reactionmixture was stirred for 30 min at 0° C. and then for 90 min at roomtemperature. After removing the solvent, the crude was precipitated inEt₂O to give compound 536 (235 mg, 0.257 mmol, 79%) as the TFA salt. ¹HNMR (D₂O, 400 MHz) δ 3.50 (t, J=6.0 Hz, 2H), 3.09-3.15 (m, 18H),2.06-2.10 (m, 6H), 1.94-1.97 (m, 2H), 1.76 (brs, 4H). Molecular weightfor C₁₆H₃₉N₈ (M+H)⁺ Calc. 343.33. Found 343.2.

Compound 537:

To a solution of compound 536 (200 mg, 0.219 mmol) in pyridine (3 mL),trifluoroacetic anhydride (0.245 mL, 1.75 mmol) was added dropwise at 0°C. The reaction mixture was stirred for 14 hours at room temperature.After removing solvent, the crude was purified by silica gel columnchromatography (Hexane:EtOAc=1:2, R_(f)=0.34) to give compound 537 (127mg, 0.154 mmol, 70%). ¹H NMR (DMSO-d₆, 400 MHz) δ 9.45-9.53 (m, 1H),3.30-3.44 (m, 12H), 3.22 (t, J=6.0 Hz, 2H), 1.80-1.89 (m, 8H), 1.55(brs, 4H). ¹⁹F NMR (376 MHz, DMSO-d₆) δ −70.90, −70.92, −70.96, −70.98,−71.06, −71.11, −71.13, −71.16, −71.20, −71.34, −77.26, −77.28, −77.41.Molecular weight for C₂₆H₃₃F₁₅N₈NaO₅ (M+Na)⁺ Calc. 845.22. Found 845.3.The oligoamine derivatives with azide functionality were synthesized asfollows.

Compound 538:

To a solution of compound 534 (40 g, 55.8 mmol) in methanol (300 mL),triethylamine (64 mL) and acrylonitrile (7.35 mL, 111.6 mmol) wereadded. The reaction was stirred for 14 hours at room temperature. Then,di-tert-butyl dicarbonate (37.1 g, 170 mmol) was slowly added at ° C.and the mixture was stirred for 14 hour at room temperature. Afteraqueous work-up, the crude was purified by silica gel columnchromatography (Hexane:EtOAc=1:4, R_(f)=0.72) to give 538 (23.9 g, 23.4mmol, 42%). ¹H NMR (DMSO-d₆, 400 MHz) δ 3.40 (t, J=6.4 Hz, 4H),3.09-3.15 (m, 20H), 2.69 (t, J=6.4 Hz, 4H), 1.36-1.65 (m, 66H). ¹³C NMR(DMSO-d₆, 100 MHz) δ□ 170.3, 154.5, 119.0, 79.1, 78.3, 78.2, 59.7, 45.0,44.1, 42.4, 28.0, 27.9, 20.7, 14.1. Molecular weight for C₅₂H₉₄N₈NaO₁₂(M+Na)⁺ Calc. 1045.69. Found 1045.5.

Compound 539:

A solution of compound 538 (7.88 g, 7.70 mmol) in methanol (80 mL) andacetic acid (10 mL) was hydrogenated over Pd(OH)₂/C (8.0 g) at 50 psihydrogen pressure for 14 hours. After filtration through Celite, thefiltrate was concentrated in vacuo. The residue was extracted with CHCl₃(300 mL) and 1 M NaOH (40 mL). The organic layer was separated, driedover anhydrous MgSO₄, filtered and concentrated to afford compound 539(7.90 g, 7.66 mmol, 99%) as a viscous oil. ¹H NMR (DMSO-d₆, 400 MHz) δ3.62 (brs, 4H), 3.08-3.14 (m, 24H), 1.36-1.63 (m, 70H). ¹³C NMR(DMSO-d₆, 100 MHz) δ□ 154.6, 154.5, 154.4, 79.1, 78.3, 78.2, 46.3, 44.3,38.5, 30.8, 28.0, 27.2, 25.1. Molecular weight for C₅₂H₁₀₃N₈O₁₂ (M+H)⁺Calc. 1031.77. Found 1031.5.

Compound 541:

To a solution of compound 539 (7.40 g, 7.18 mmol) in methanol (72 mL),triethylamine (2.5 mL, 18.0 mmol), copper sulfate pentahydrate (18 mg,0.072 mmol) and imidazole-1-sulfonyl azide hydrochloride (903 mg, 4.31mmol) were added. The reaction mixture was stirred for 2 hours at roomtemperature (540; Molecular weight for C₅₂H₁₀₁N₁₀O₁₂ (M+H)⁺ Calc.1057.76. Found 1057.5). Then, ethyl trifluoroacetate (2.60 mL, 21.5mmol) was added and the mixture was stirred for 2 hours at roomtemperature. After aqueous work-up, the crude was purified by silica gelcolumn chromatography (Hexane:EtOAc=1:1, R_(f)=0.38) to give 541 (1.75g, 1.52 mmol, 21%). ¹H NMR (DMSO-d₆, 400 MHz) δ 9.38 (brs, 1H),3.10-3.33 (m, 28H), 1.31-1.71 (m, 70H). ¹⁹F NMR (376 MHz, DMSO-d₆) δ−77.27. ¹³C NMR (DMSO-d₆, 100 MHz) δ□ 156.3, 155.9, 154.5, 154.4, 120.2,117.3, 114.5, 78.4, 78.3, 78.2, 63.4, 48.4, 46.3, 44.2, 37.0, 27.9,27.3, 25.1. Molecular weight for C₅₄H₉₉F₃N₁₀NaO₁₃ (M+Na)⁺ Calc. 1175.72.Found 1175.3.

Compound 542:

To a solution of compound 541 (1.75 g, 1.52 mmol) in CH₂Cl₂ (40 mL),trifluoroacetic acid (10 mL) was slowly added at 0° C. The reactionmixture was stirred for 1 hour at 0° C. and then for 6 hours at roomtemperature. Removal of the solvent in vacuo afforded compound 542 as awhite foam. This material was used for the next reaction without furtherpurification. Molecular weight for C₂₄H₅₂F₃N₁₀O (M+H)⁺ Calc. 553.43.Found 553.3.

Compound 543:

To a solution of compound 542 (1.52 mmol) in pyridine (20 mL),trifluoroacetic anhydride (2.11 mL, 15.2 mmol) was added dropwise at 0°C. The reaction mixture was stirred for 14 hours at room temperature.After removing solvent, the crude was extracted with CH₂Cl₂ and H₂O. Theorganic layer was dried over anhydrous Na₂SO₄, then filtered andconcentrated in vacuo. The crude was purified by silica gel columnchromatography (EtOAc) to give compound 543 (1.45 g, 1.28 mmol, 85%). ¹HNMR (DMSO-d₆, 400 MHz) δ 9.43-9.51 (m, 1H), 3.32-3.38 (m, 26H),3.21-3.23 (m, 2H), 1.80-1.87 (m, 12H), 1.55 (brs, 4H). ¹⁹F NMR (376 MHz,DMSO-d₆) δ −71.00, −71.05, −71.13, −71.21, −71.26, −71.32, −71.35,−71.40, −77.32, −77.35, −77.47. ¹³C NMR (DMSO-d₆, 100 MHz) δ 156.5,155.8, 155.6, 155.5, 155.3, 120.5, 117.7, 114.8, 114.4, 111.9, 48.3,47.9, 46.7, 45.9, 44.6, 44.2, 44.0, 43.7, 36.9, 36.4, 27.7, 27.4, 25.9,25.7, 25.3, 25.0, 24.0, 23.5, 23.3. Molecular weight for C₃₆H₄₄F₂₁N₁₀O₇(M−H)⁻ Calc. 1127.31. Found 1127.0.

Example 12 Preparation of 1′-Triazoylribofuranose Derivatives

Example 13 Functionalized Peptides for Conjugation to Oligonucleotides

The peptides shown in Table 5 are synthesized by following solid phaseFmoc chemistry for conjugation to oligonucleotides via the clickchemistry approach. Peptides P1-P3 contains a azido moiety and peptidesP4-P6 contain terminal alkynes to conjugate to oligonucleotides or acarrier molecules with complementary function group.

TABLE 5 Functionalized peptide for conjugation Functional group forPeptide conjugation P1N₃-(CH₂)₁₅-CO-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg- AzideArg-Pro-Pro-Gln-NH₂ P2 cyclo-[Phe-Arg-Gly-Asp-Lys(N₃-(CH₂)₁₅-COOH)]Azide P3 N₃-(CH₂)₁₅-CO-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-NH₂ Azide P4C═C-(CH₂)₃-CO-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg- AlkyneArg-Pro-Pro-Gln-NH₂ P5 cyclo-[Phe-Arg-Gly-Asp-Lys(C═C-(CH₂)₃-COOH)]Alkyne P6 C═C-(CH₂)₃-CO-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Alkyne NH₂

Example 14 On Support Post Synthetic Click Chemistry afterOligonucleotide Synthesis

Scheme 39 shows exemplary on support conjugation of ligands tooligonucleotide by click chemistry. One equivalent ofoligonucleotide-alkyne (1 μmole scale synthesis0 was reacted with 10equivalent of C22-azide (MeOH/THF 1:1 v/v) in the presence of 0.25equivalent CuSO₄ and 2 equivalents of sodium ascorbate. The resultingconjugated oligonucleotide (33561) was analyzed by HPLC and results areshown in FIG. 12. Conjugated oligonucleotide was purified by RP-HPLC towith 88% purity as determined by HPLC analysis, FIG. 13.

Example 15 Post Synthetic Labeling of Oligonucleotides with6-Carboxyfluorescein by Click Chemistry a. Synthesis of6-Carboxyfluorescein-propargylamide (Alkynyl FAM)

A solution of 6.8 ul (0.1 mmol) of propargylamine in DMF (1.0 ml) wasadded to a solution of 22 mg (0.046 mmol) of 6-carboxyfluorescein NHSester in DMF (1.0 ml) and 0.1 M NaHCO3 (0.2 ml) buffer. After overnightstirring at room temperature, the solvent was removed under vacuum. Asmall amount of reaction mixture was submitted for LC-MS analysis. Thecrude mixture was purified by Silica gel column chromatography. (DCM:MeOH 9:1). LC-MS analysis confirms that alkenyl FAM is formed. Cal Mass:414.09. Found Mass: 414.0.

b. Synthesis of Azio-Labeled Oligonucleotide

A schematic representation of an azido-labeled oligonucleotide is shownin FIG. 14. To incorporate the azido group at the 3′-end of siRNA, 100nmole of Amino modified siRNA (A1-3991) in 100 ul of 0.2 M NaHCO₃ buffer(pH 9.0) incubated for 15 h at room temperature with 2.0 umole of azidoester in 25 ul DMF. After 15 h sample was analyze by IEX analysis. Leavethe sample at room temperature for 9 h to complete the reaction. After24 h sample was analyzed by LC-MS and it confirms the integrity of thecompound. Cal Mass: 7269.35. Found Mass: 7268.56.

c. Click Chemistry

FIG. 15 shows 6-Carboxyfluorescein-propargylamide conjugated with anazido-labeled oligonucleotide by Click chemistry. Azido-oligonucleotide(5.0 nmol) in 100 ul of water was reacted with a 150-fold excess ofalkynyl FAM in 25 ul of dry DMSO at 80° C. for 72 h. Unreacted dye wasremoved by size-exclusion chromatography on a PD-10 column. LC-MS datashows that 1, 3-dipolar cycloaddition between alkynyl6-carboxyfluorescein (FAM) and azido-labeled single-stranded RNA wascarried out under aqueous conditions to produce FAM-labeled siRNA, FIG.16. Fluorescein-conjugated oligomer (n): Cal Mass: 7684. Found Mass7682.3, unconjugated starting material (b): Cal Mass: 7269. Found Mass7277.

Example 16 Azide Modified C6-Aminohydroxyprolinol

The DMTr-protected C6-aminoprolinol were converted to the correspondingazide derivative as follows.

Compound 545:

To a solution of compound 544 (200 mg, 0.375 mmol) in methanol (5 mL),triethylamine (0.157 mL, 1.13 mmol), copper sulfate pentahydrate (1 mg,0.00375 mmol), and imidazole-1-sulfonyl azide hydrochloride (94 mg, 0.45mmol) were added. The reaction mixture was stirred for 1 hour at roomtemperature. The product formation was confirmed by mass analysis.Molecular weight for C₃₂H₃₈N₄NaO₅ (M+Na)⁺ Calc. 581.27. Found 581.0;C₃₂H₃₉N₄O₅ (M+H)⁺ Calc. 559.29. Found 559.0.

Example 17 Triazole-Linked Nucleoside Building Block

Compound 547:

To a solution of compound 546 (Prime organics, 2.13 g, 7.49 mmol) inpyridine (30 mL), phenylchloroformate (1.04 mL, 8.24 mmol) was added at0° C. After stirring for 2 hours at room temperature, propargylamine wasadded at 0° C. The mixture was stirred for 14 hours at room temperature.After removal of the solvent, aqueous work-up and purification by silicagel column chromatography (Hexane:EtOAc=1:2, R_(f)=0.19) to give 547(2.73 g, 7.47 mmol, 99%). ¹H NMR (DMSO-d₆, 400 MHz) δ 11.37 (s, 1H),7.88 (t, J=5.6 Hz, 1H), 7.46 (s, 1H), 6.19 (dd, J=5.8 Hz, 9.0 Hz, 1H),5.17-5.18 (m, 1H), 4.23-4.26 (m, 1H), 4.10-4.15 (m, 2H), 3.81 (dd, J=2.4Hz, 5.6 Hz, 2H), 3.13 (t, J=2.4 Hz, 1H), 2.43-2.47 (m, 1H), 2.24 (dd,J=5.8 Hz, 13.8 Hz, 1H), 2.07 (s, 3H), 1.79 (s, 3H). ¹³C NMR (DMSO-d₆,100 MHz) δ 170.0, 163.6, 155.5, 150.5, 135.6, 109.9, 83.7, 81.4, 81.1,74.3, 73.2, 64.2, 35.6, 29.8, 12.2. Molecular weight for C₁₆H₁₆N₃NaO₇(M+Na)⁺ Calc. 388.11. Found 388.0.

Compound 548:

To a solution of compound 547 (2.59 g, 7.09 mmol) and 3′-azidothymidine(1.89 g, 7.09 mmol) in THF (50 mL) and MeOH (50 mL), H₂O (25 mL),(+)-sodium L-ascorbate (140 mg, 0.709 mmol), and copper sulfatepentahydrate (35 mg, 0.142 mmol) were added. The reaction mixture washeated at 70° C. for 2 hours. After removal of the solvent,co-evaporation with pyridine twice gave pale-yellow foam (quantitativeyield). The material was used for the next reaction. Analytical sampleswere obtained by silica gel column chromatography (10% MeOH in CH₂Cl₂,R_(f)=0.36). ¹H NMR (DMSO-d₆, 400 MHz) δ 11.38 (s, 1H), 11.37 (s, 1H),8.16 (s, 1H), 7.97 (t, J=5.6 Hz, 1H), 7.81 (d, J=0.8 Hz, 1H), 7.46 (d,J=1.2 Hz, 1H), 6.41 (t, J=6.6 Hz, 1H), 6.19 (dd, J=6.0 Hz, 8.8 Hz, 1H),5.33-5.37 (m, 1H), 5.29 (t, J=5.2 Hz, 1H), 5.18 (d, J=6.8 Hz 1H),4.09-4.29 (m, 6H), 3.66-3.71 (m, 1H), 3.57-3.62 (m, 1H), 2.61-2.73 (m,2H), 2.44-2.46 (m, 1H), 2.20-2.25 (m, 1H), 2.07 (s, 3H), 1.81 (s, 3H),1.72 (s, 3H). ¹³C NMR (DMSO-d₆, 100 MHz) δ 170.1, 163.7, 163.6, 155.9,149.6, 145.2, 136.3, 135.9, 124.0, 122.7, 110.0, 109.8, 84.7, 84.0,83.9, 81.6, 74.6, 64.2, 60.7, 59.4, 47.6, 37.2, 35.9, 35.6, 20.8, 12.2,12.1. Molecular weight for C₂₆H₃₂N₈NaO₁₁ (M+Na)⁺ Calc. 655.21. Found655.0.

Compound 549:

To a solution of compound 548 (-7.09 mmol) in pyridine (70 mL), DMTrCl(2.76 g, 8.15 mmol) was added. The reaction mixture was stirred at roomtemperature for 14 hours. After removal of the solvent, aqueous work-upand purification by silica gel column chromatography (5% MeOH in CH₂Cl₂,R_(f)=0.26) to give 549 (5.69 g, 6.09 mmol, 86%). ¹H NMR (DMSO-d₆, 400MHz) δ 11.38 (s, 2H), 8.17 (s, 1H), 7.97 (t, J=5.8 Hz, 1H), 7.66 (s,1H), 7.46 (s, 1H), 7.21-7.35 (m, 9H), 6.86-6.87 (m, 4H), 6.42 (t, J=6.4Hz, 1H), 6.20 (dd, J=6.0 Hz, 8.8 Hz, 1H), 5.54 (dd, J=7.2 Hz, 14.4 Hz,1H), 5.19 (d, J=6.0 Hz, 1H), 4.10-4.35 (m, 6H), 3.73 (s, 6H), 3.26-3.34(m, 2H), 2.73-2.77 (m, 2H), 2.44-2.46 (m, 1H), 2.21-2.26 (m, 1H), 2.06(s, 3H), 1.71 (s, 3H), 1.59 (s, 3H). ¹³C NMR (DMSO-d₆, 100 MHz) δ 170.0,163.6, 155.8, 150.4, 145.2, 144.5, 135.7, 135.1, 126.7, 122.2, 113.2,109.9, 109.8, 85.9, 83.8, 83.8, 82.2, 81.4, 74.4, 64.1, 63.0, 59.2,55.0, 37.2, 35.9, 35.5, 20.8, 12.1, 11.8. Molecular weight forC₄₇H₄₉N₈O₁₃ (M−H)⁻ Calc. 933.34. Found 933.0.

Compound 550:

To a solution of compound 549 (5.48 g, 5.86 mmol) in CH₂Cl₂ (90 mL) andMeOH (10 mL), 0.5 M NaOMe in MeOH (22.8 mL, 11.4 mmol) was added. Thereaction mixture was stirred at room temperature for 14 hours. Afterremoval of the solvent, the crude was purified by silica gel columnchromatography (10% MeOH in CH₂Cl₂, R_(f)=0.46) to give 550 (4.59 g,5.14 mmol, 88%). ¹H NMR (DMSO-d₆, 400 MHz) δ 11.39 (s, 1H), 11.30 (s,1H), 8.15 (s, 1H), 7.87 (t, J=5.8 Hz, 1H), 7.65 (s, 1H), 7.42 (s, 1H),7.20-7.34 (m, 9H), 6.85-6.87 (m, 4H), 6.40 (t, J=6.6 Hz, 1H), 6.18-6.21(m, 1H), 5.52 (dd, J=7.2 Hz, 14.4 Hz, 1H), 5.37 (d, J=4.4 Hz, 1H),4.20-4.32 (m, 6H), 4.03-4.06 (m, 1H), 3.91-3.93 (m, 1H), 3.73 (s, 6H),3.06-3.27 (m, 1H), 2.72-2.76 (m, 2H), 2.19-2.26 (m, 1H), 2.03-2.07 (m,1H), 1.71 (s, 3H), 1.59 (s, 3H). ¹³C NMR (DMSO-d₆, 100 MHz) δ 163.7,163.6, 156.0, 150.4, 145.3, 144.5, 135.9, 135.1, 126.8, 122.2, 113.2,109.8, 109.7, 85.9, 84.2, 83.8, 82.2, 70.4, 63.0, 59.2, 55.0, 38.6,37.2, 35.9, 12.1, 11.8. Molecular weight for C₄₅H₄₇N₈O₁₂ (M−H)⁻ Calc.891.33. Found 891.0.

Compound 551:

To a solution of compound 550 (3.56 g, 3.99 mmol) in CH₂Cl₂ (75 mL),DMAP (1.46 g, 12.0 mmol) and succinic anhydride (799 mg, 7.98 mmol) wereadded. The reaction mixture was stirred overnight at room temperature.Purification by silica gel column chromatography (5% MeOH/5% Et₃N inCH₂Cl₂, R_(f)=0.11) of the crude mixture gave the compound 551 as thecorresponding triethylammonium salt (4.29 g, 3.92 mmol, 98%). ¹H NMR(DMSO-d₆, 400 MHz) δ 11.34 (brs, 2H), 8.17 (s, 1H), 7.98 (t, J=5.8 Hz,1H), 7.65 (s, 1H), 7.45 (s, 1H), 7.20-7.34 (m, 9H), 6.84-6.86 (m, 4H),6.40 (t, J=6.4 Hz, 1H), 6.19 (dd, J=6.0 Hz, 8.8 Hz, 1H), 5.53 (dd, J=7.0Hz, 14.6 Hz, 1H), 5.18 (d, J=6.0 Hz, 1H), 4.08-4.32 (m, 6H), 3.72 (s,6H), 3.23-3.39 (m, 2H), 2.72-2.76 (m, 2H), 2.41-2.54 (m, 5H), 2.19-2.24(m, 1H), 1.70 (s, 3H), 1.58 (s, 3H). ¹³C NMR (DMSO-d₆, 100 MHz) δ 173.7,172.1, 163.7, 163.6, 155.8, 150.4, 145.2, 144.5, 135.7, 135.1, 126.7,122.2, 113.2, 109.8, 85.9, 83.8, 82.2, 81.4, 74.4, 64.2, 63.0, 62.5,59.2, 52.0, 37.2, 35.8, 35.5, 29.4, 29.2, 12.1, 11.8. Molecular weightfor C₄₉H₅₁N₈O₁₅ (M−H)⁻ Calc. 991.35. Found 991.0.

Compound 552:

Compound 551 (4.21 g, 3.85 mmol) was dissolved in DMF (250 mL). HBTU(1.53 g, 4.04 mmol) then iPr₂NEt (3.35 mL, 19.3 mmol) and finallyCPG-NH₂ (Prime Synthesis CPG-585 angstrom, NH₂ loading=124 μmol/g) (34.2g, 4.24 mmol) were added in succession. The mixture was shaken for 4 hat room temperature, then the solid was collected by filtration, washedwith CH₂Cl₂ (500 mL), then 50% MeOH/CH₂Cl₂ (500 mL) and dried in vacuo.The residual amino groups were capped by shaking for 1 h withAc₂O/Pyridine/Et₃N (80 mL/240 mL/16 mL). Filtration and washing withCH₂Cl₂ (500 mL) and 50% MeOH/CH₂Cl₂ (500 mL) then drying overnight invacuo gave compound 552 (˜36 g, 64 μmol/g).

Example 18 Conjugation of Free Spermine Molecule to RNA on the SolidSupport Via Click Chemistry

Compound 554: (1) RNA alkyne on the solid support, A-33072.1, (11 mg,0.62 μmol) was treated with spermine azide (553, 3.53 mg, 6.18 μmol) inthe presence of CuSO₄.5H₂O (0.62 μmol) and sodium ascorbate (3.1 μmole),and the mixture was kept on a heating block at 65° C. for 2 h. Thesolvent volume of the reaction was maintained as a 1:1 ratio oft-BuOH/H₂O (v/v) (1 ml). After reaction the beads were subsequentlywashed with water, methanol and acetonitrile and allowed to dry at roomtemperature. (2) Deprotection: The reacted beads were treated withaqueous methylamine (125 μl) for 20 min at 65° C., then cooled to −20°C. for 20 min. The filtrate was collected, and the beads were washedwith DMSO (100 μl×3) and combined them with the filtrate, then cooled at−20° C. for 10 min. Triethylamine trihydrofluoride (175 μl) was added tothe combined filtrate and the mixture was kept at 65° C. for 20 minAfter dialysis, the crude reaction mixture was analyzed by reverse-phaseHPLC and LC-MS, FIG. 17. (3) RP-HPLC and LC-MS: The material was loadedto an analytical RP-HPLC on a gradient using two buffer systems (0.1MTEAA, pH 7.0 and acetonitrile), % B=0-40%/20 min, ˜100%/30 min, 1ml/min, linear gradient, UV: 260 nm; Column: XTerra RP-18, 4.6×250 mm.The crude materials also analyzed by LC-MS (calcd mass 7220. found 7219and 7260).

Example 19 Conjugation of Boc-Protected Spermine Molecule to RNA on theSolid Support Via Click Chemistry

Compound 555: The reaction between boc-protected spermine azide 531a andRNA alkyne A-33072.1 on the solid support was described below.Boc-protected spermine azide was used to establish a model reaction forclick chemistry with the RNA alkyne.

(1) RNA alkyne on the solid support, A-33072.1, (11 mg, 0.62 μmol) wastreated with boc-protected spermine azide (531a, excess) in the presenceof CuSO₄.5H₂O (0.93 μmol) and sodium ascorbate (3.1 μmole), and themixture was kept on a heating block at 50° C. for 12 h. The volume ofthe solvent for the reaction was maintained as a 1:1 ratio of t-BuOH/H₂O(v/v) (1 ml). After reaction the beads were subsequently washed withwater, methanol and acetonitrile and allowed to dry at room temperature.

(2) Deprotection: The reacted beads were treated with aqueousmethylamine (125 μl) for 20 min at 65° C., then cooled to −20° C. for 20min. The filtrate was collected, and the beads were washed with DMSO(100 μl×3) and combined them with the filtrate, then cooled at −20° C.for 10 min. Triethylamine trihydrofluoride (175 μl) was added to thecombined filtrate and the mixture was kept at 65° C. for 20 min. Afterdialysis, the crude reaction mixture was analyzed by reverse-phase HPLCand LC-MS.

(3) RP-HPLC and LC-MS: The material was loaded to an analytical RP-HPLCon a gradient using two buffer systems (0.1M TEAA, pH 7.0 andacetonitrile), % B=0-60%/20 min, ˜100%/30 min, 1 ml/min, lineargradient, UV: 260 nm; Column: XTerra RP-18, 4.6×250 mm. The crudematerials also analyzed by LC-MS (calcd mass 7520. found 7519), FIG. 18.

Example 20 Conjugation of TFA-Protected Spermine Molecule to RNA on theSolid Support Via Click Chemistry

Compound 556: The reaction between TFA-protected spermine azide 533 andRNA alkyne A-37068.1 on the solid support was described below.

(1) RNA alkyne on the solid support, A-37086.1, (11 mg, 1 μmol) wastreated with TFA-protected spermine azide (533, excess) in the presenceof CuSO₄.5H₂O (1.5 μmol) and sodium ascorbate (5 μmole), and the mixturewas kept on a heating block at 50° C. for overnight. The volume of thereaction solvent was maintained as a 1:1 ratio of t-BuOH/H₂O (v/v) (1ml). After reaction the beads were subsequently washed with water,methanol and acetonitrile and allowed to dry at room temperature.

(2) Deprotection: The reacted beads were treated with aqueousmethylamine (125 μl) for 20 min at 65° C., then cooled to −20° C. for 20min. The filtrate was collected, and the beads were washed with DMSO(100 μl×3) and combined them with the filtrate, then cooled at −20° C.for 10 min. Triethylamine trihydrofluoride (175 μl) was added to thecombined filtrate and the mixture was kept at 65° C. for 20 min.

(3) LC-MS: After dialysis, the crude reaction mixture was analyzed byLC-MS (calcd mass 7235. found 7234).

Example 21 Conjugation of TFA-Protected Spermine (Seven Amines) to RNAon the Solid Support Via Click Chemistry

Compound 557 can be synthesized by a procedure similar to that ofexample 19 or 20 and utilizing the extended spermine 543.

Example 22 Oligonucleotides

1. Synthesis of Single-Stranded RNA by Solid Phase Method

RNA oligonucleotides (Table 6) were synthesized on a DNA synthesizer ABI394 using standard phosphoramidite chemistry with commercially available5′-O-(4,4′-dimethoxytrityl)-3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramiditemonomers of uridine (U), 4-N-benzoylcytidine (C^(Bz)),6-N-benzoyladenosine (A^(Bz)) and 2-Nisobutyrylguanosine (G^(iBu)) with2′-O-t-butyldimethylsilyl protected phosphoramidites, and5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine-3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite(T). Modified RNA phosphoramidites and CPG used in this study are shownin FIGS. 19 and 20. After cleavage and de-protection, RNAoligonucleotides were purified by anion-exchange or reverse phasehigh-performance liquid chromatography (AX-HPLC or RP-HPLC) andcharacterized by LC-MS.

2. Synthesis of RNA-Alkyne Scaffolds

The solid-supported phosphotriester alkyne scaffolds (3′, 5′ terminalpositions and internal position in Table 7) were synthesized in the sameway as what is mentioned in the previous section except for extendednucleotide coupling time (30 min) wherever alkyne monomer was involved.After synthesis, a small portion of CPG was deprotected and analyzed byAX-HPLC and LC-MS.

3. Cu(I)-Catalyzed 1,3-Dipolar Cycloaddition Reaction Optimization

Click reaction conditions were optimized using a 5′-alkyne modified RNAon CPG (seq. #33072, Table 8). The effects of equivalence of statingmaterials, temperature and solvent were investigated in the clickreaction of seq. #33072 with 1-azido-docosane (C22-azide) (Scheme 39).The optimization results were synthesized in Table 9. It would foundthat TBTA can facilitate the click reaction in the conditions used inthis study. The click did not happen without TBTA (reaction No. 1 and2). Cu²⁺ level should maintain at a certain level (0.4 eqv. of alkyne inreaction No. 5) and too low Cu²⁺ level negatively affect the clickreaction (reaction No. 9). It was also found that DMF, DMSO could beused as a co-solvent but they did not show as good as THF (reaction No.5, 7, 8). Microwave aided reaction was much faster. The reaction wascompleted after two days. This mild condition may be useful for somethermally unstable RNA sequences (reaction No. 12-13). In summary, theoptimal conditions we found in the click reactions of RNA-alkyne withC22-azide include the molar ratio of RNA-alkyne:azide:Cu:TBTA:NaAsc(1:2.9:0.4:2.8:3.2), microwave-aided heating at 60° C. for 45 min inH₂O/MeOH/THF (8/8/5 v/v/v).

After reaction optimization, different azides (FIG. 21) were tested for1,3-dipolar cycloaddition with this RNA-alkyne in the optimizedconditions. It was shown that all click reactions gave excellentreaction completion rate except for 1-azido-adamantane with only 50%completion, Table 9. Its steric environment was expected to account forthis slow click reaction. Extended reaction to 90 min did not improvethe reaction completion rate (data not shown).

4. General Procedure for Cu(I)-Catalyzed 1,3-Dipolar Cycloaddition

To a solid-supported oligoalkyne phosphotriester seq. #33072-37088 inTable 8 (0.62 μmol) was added a mixture of an azide (FIG. 21) (3 equivby alkyne, 1.8 μmol, 36 μL of a 50 mM solution in THF), CuSO₄ (0.4equiv, 0.25 μmol, 5 μL of a 50 mM solution in H₂O), freshly preparedsodium ascorbate (3.2 equiv, 2.0 μmol, 12 μL of a 50 mM solution inH2O), TBTA (2.8 equiv, 1.7 μmol, 35 μL of a 50 mM solution in THF).Water, MeOH and THF were added to obtain a total volume of 1200 μL(around 8:8:5 v/v/v). The resulting preparation was heated in a sealedglass tube with Explorer-48 (CEM) microwave synthesizer at a 100 W and a30 s premixing time. The temperature was monitored with an internalinfrared probe to 60° C. during 45 min. The solution was removed, andthe CPG supports were washed with THF and MeOH then dried. Aftercleavage and de-protection, the click reaction progress was analyzed byRP-HPLC and the products were characterized by LC-MS.

The click reaction mixtures were purified by RP-HPCL usingsemi-preparative DeltaPak C4 column (Waters) with 0-90% ACN (buffer B)gradient in 40 min. The purified RNAs were analyzed by RP-HPCL andLC-MS. The HPLC profiles of purified click product were shown in FIG.23.

Table 11 summarizes the click modified oligonucleotides that weresynthesized.

TABLE 6 RNA and modified RNA sequences made by solid phase sequencesStrand Strand Calc. Obs. Yield No. (S/AS) Sequence (5′-3′) mass mass(OD) Purity  1000 S           CUUACGCUGAGUACUUCGATT 6607.0 — stock — 1001 AS           UCGAAGUACUCAGCGUAAGTT 6693.1 — stock — 33024.1 S      C(Uda)UACGCUGAGUACUUCGATT 6915.6 6914 53.0 93% 33025.1 S      CU(Uda)ACGCUGAGUACUUCGATT 6915.6 108.0 92% 33026.1 S      CUUACGC(Uda)GAGUACUUCGATT 6915.6 229.0 91% 33027.1 S      CUUACGCUGAG(Uda)ACUUCGATT 6915.6 110.0 95% 33028.1 S      CUUACGCUGAGUAC(Uda)UCGATT 6915.6 200.0 97% 33029.1 S      CUUACGCUGAGUACU(Uda)CGATT 6915.6 213.0 96% 33031.1 S    CUUACGCUGAGUACUUCGAT(Uda2p) 6917.5 72.0 93% 33032.1 SCUUACGCUGAG(Uda)ACUUCGAT(Uda2p) 7226.1 68.0 88% 33033.1 SC(Uda)UACGCUGAGUACUUCGAT(Uda2p) 7226.1 79.0 86% 33035.1 AS    UCGAAGUACUCAGCGUAAGT(Uda2p) 7003.6 36.0 86% 33034.1s S       Q74CUUACGCUGAGUACUUCGATT 7285.7 59.0 93% 33071.1 S       Q75CUUACGCUGAGUACUUCGATT 7328.5 37.0 92% Note: (Uda):2′-docosanoyl-uridine-3′-phosphate (Uda2p):3′-docosanoyl-uridine-2′-phosphate (amidite and CPG) Q74:uridine-5′-(N1-linolyl-4-methylaminocarbonyl-1,2,3 triazol)-3′-phosphateQ75: uridine-5′-[N1-(GalNAc-1-ethyloxyethyl)-1,2,3 triazol]-3′-phosphate

TABLE 7 RNA-alkyne scaffolds made for click reactions in this studyStrand Calc. Obs. Crude No. Sequence (5′-3′) mass mass purity¹ 33072.1   Q83CUUACGCUGAGUACUUCGAdTdT 6994.2 6992 62.3% 36594.1  AAcGcuGGGcGuuAAucAAdTdTL123 7191.7 7190 68.8% 37074.1 CUUACGCUGAGUACUUCGAdTdT(Tpy) 6965.2 6964 61.2% 37076.1CUUACGCUGAGUACUUCGAdTdT(T3py) 6965.2 6964 58.7% 37078.1  CUUACGCUGAG(Tpy)ACUUCGAdTdT 6659.1 6657 46.9% 37080.1 CUUACGCUGAG(T3py)ACUUCGAdTdT 6659.1 6658 58.9% 37082.1 (Tpy)CUUACGCUGAGUACUUCGAdTdT 6965.23 6964 43.5% 37084.1(T3py)CUUACGCUGAGUACUUCGAdTdT 6965.23 6963 68.0% 37086.1(u5py)CUUACGCUGAGUACUUCGAdTdT 7009.26 7006 68.0% 37088.1(u5pe)CUUACGCUGAGUACUUCGAdTdT 7037.32 7034 67.3% ¹Purity was measured byRP-HPLC (DeltaPak C4 column, 150 × 3.9 mm I.D., 5 μm, 300 Å; buffer A:50 mM TEAA, pH 7.0; buffer B: ACN; gradient: 0-70% buffer B in 24 min;30° C., 1 mL/min)

TABLE 8 Cu(I)-Catalyzed 1,3-Dipolar Cycloaddition Reaction Optimization(RNA-alkyne seq#33072 clicks with C22 azide in Scheme 39) Startingmaterial (molar ratio of Rxn alkyne:azide:Cu: Temp. (° C.) ReactionRight No. TBTA:NaAsc) Solvent Time (min) completion (%) mass?  11:16:0.4:0:3.2 H₂O/MeOH/THF 60° C.; 45 min No rxn No  2 1:16:0.4:2.8:3.2H₂O/MeOH/THF 60° C.; 45 min   70.8% ¹ Yes  3 1:8.6:0.4:2.8:3.2H₂O/MeOH/THF 60° C.; 45 min 98.1% Yes  4 1:5.7:0.4:2.8:3.2 H₂O/MeOH/THF60° C.; 45 min ~100%  Yes  5 1:2.9:0.4:2.8:3.2 H₂O/MeOH/THF 60° C.; 45min 98.9% Yes  6 1:2.9:0.1:2.8:3.2 H₂O/MeOH/THF 60° C.; 45 min 49.4% Yes 7 1:2.9:0.4:2.8:3.2 H₂O/MeOH/DMSO 60° C.; 45 min 88.3% Yes  81:2.9:0.4:2.8:3.2 H₂O/MeOH/DMF 60° C.; 45 min 78.5% Yes    9 ²1:2.9:0.4:2.8:3.2 H₂O/MeOH/DMF 60° C.; 45 min 56.5% Yes (10× dilution)  10 ³ 1:2.9:0.4:2.8:3.2 H₂O/MeOH/DMF 60° C.; 45 min 59.7% Yes (10×concentration) 11 1:2.9:0.4:2.8:3.2 H₂O/MeOH/THF 60° C.; 5 min 57.0% Yes12 1:2.9:0.4:2.8:3.2 H₂O/MeOH/THF r.t., 17 hr 88.4% Yes 131:2.9:0.4:2.8:3.2 H₂O/MeOH/THF r.t., 48 hr ~100%  Yes ¹ Reactioncompletion was measured by RP-HPLC (DeltaPak C4 column, 150 × 3.9 mmI.D., 5 μm, 300 Å; buffer A: 50 mM TEAA, pH 7.0; buffer B: ACN;gradient: 0-70% in 24 min; 30° C., 1 mL/min); ² azide concentration inreaction solution was 14.9 mM; ³ azide concentration in reactionsolution was 0.5 mM;

TABLE 9 Cu(I)-Catalyzed 1,3-Dipolar Cycloaddition with Different AzidesStrand Reaction Calc. Obs. Azides No. Sequences completion (%)¹ massmass 1-azido-docosane 33561.1 Q87CUUACGCUGAGUACUUCGAdTdT 98.1% 7345.87344 (C22-azide) 1-azido-octadeca- 33034.1c Q74CUUACGCUGAGUACUUCGAdTdT93.4% 7285.7 7284 6,9-diene (C18(ω = 2)-azide) 1-azido-octadec-9-33559.1 Q85CUUACGCUGAGUACUUCGAdTdT 88.9% 7287.7 7285 diene (C18(ω = 1)-azide) 1-azido-octadecane 33560.1 Q86CUUACGCUGAGUACUUCGAdTdT 95.3%7289.7 7288 (C18-azide) 1-azido-adamantane 33562.1Q88CUUACGCUGAGUACUUCGAdTdT 50.6% 7171.5 7169 Cholesterol-azide 33563.1Q89CUUACGCUGAGUACUUCGAdTdT 95.7% 7549.1 7547 ¹Reaction completion wasmeasured by RP-HPLC (DeltaPak C4 column, 150 × 3.9 mm I.D., 5 μm, 300 Å;buffer A: 50 mM TEAA, pH 7.0; buffer B: ACN; gradient: 0-70% buffer B in24 min; 30° C., 1 mL/min);

TABLE 10Cu(I)-Catalyzed 1,3-Dipolar Cycloaddition Reaction of Different RNA-alkynes with 1-Azido-octadeca-6,9-diene (C18(ω = 2)-azide (FIG. 22)RNA-alkyne Reaction Calc. Obs. position Strand No. Sequences (5′-3′)completion (%) mass mass 3′-terminal 38103.1   AAcGcuGGGcGuuAAucAAdTdTL125 51.7%¹ 7483.2 7482 37075.1  CUUACGCUGAGUACUUCGAdTdT(T1y) ~100% 7256.7 7255 37077.1 CUUACGCUGAGUACUUCGAdTdT(T31y) 90.3% 7256.7 7255 5′-terminal 33561.1    Q87CUUACGCUGAGUACUUCGAdTdT 98.1% 7345.8 7344 37083.1  (T1y)CUUACGCUGAGUACUUCGAdTdT ~100% 7256.7 7255 37085.1 (T31y)CUUACGCUGAGUACUUCGAdTdT 96.6% 7256.7 7256 37087.1(u51y1)CUUACGCUGAGUACUUCGAdTdT 91.0% 7300.7 7299 37089.1(u51y2)CUUACGCUGAGUACUUCGAdTdT 84.0% 7328.8 7326 Internal 37079.1   CUUACGCUGAG(Tly)ACUUCGAdTdT ~100% 6950.5 6949 37081.1  CUUACGCUGAG(T31y)ACUUCGAdTdT 91.5% 6950.5 6949 ¹The reactioncompletion reached ~100% if the molar ratio ofalkyne:azide:Cu:TBTA:NaAsc was 1:5.8:0.8:5.6:6.4 with other conditionsunchanged.

TABLE 11 Overall purified click products obtained in this study StrandCalc. Obs. Yield No. Sequence (5′-3′) mass mass (OD) Purity 33034.1c¹    Q74CUUACGCUGAGUACUUCGAdTdT 7285.7 7284 9.4 94% 33559.1    Q85CUUACGCUGAGUACUUCGAdTdT 7287.7 7285 10.6 94% 33560.1    Q86CUUACGCUGAGUACUUCGAdTdT 7289.7 7288 8.1 94% 33561.1    Q87CUUACGCUGAGUACUUCGAdTdT 7345.8 7344 15.5 89% 33562.1    Q88CUUACGCUGAGUACUUCGAdTdT 7171.5 7169 4.04 97% 33563.1    Q89CUUACGCUGAGUACUUCGAdTdT 7549.1 7547 24.1 95% 38103.1   AAcGcuGGGcGuuAAucAAdTdTL125 7483.2 7482 45.4 87% 37075.1  CUUACGCUGAGUACUUCGAdTdT(T1y) 7256.7 7255 46.2 96% 37077.1 CUUACGCUGAGUACUUCGAdTdT(T31y) 7256.7 7255 71.0 90% 37079.1   CUUACGCUGAG(T1y)ACUUCGAdTdT 6950.5 6949 6.3 80% 37081.1  CUUACGCUGAG(T31y)ACUUCGAdTdT 6950.5 6949 35.5 84% 37083.1  (T1y)CUUACGCUGAGUACUUCGAdTdT 7256.7 7255 20.0 79% 37085.1 (T31y)CUUACGCUGAGUACUUCGAdTdT 7256.7 7256 44.0 89% 37087.1(u51y1)CUUACGCUGAGUACUUCGAdTdT 7300.7 7299 36.0 89% 37089.1(u51y2)CUUACGCUGAGUACUUCGAdTdT 7328.8 7326 11.7 90% ¹33034.1c wasobtained by click reaction with exact sequence as 33034-1s which wasobtained by solid phase synthesis (see Table 1);

5. 3′-Exo Nuclease Stability Assays

Nuclease stability of the oligonucleotides can be determined easilyusing methods and protocols known in the art and available to one ofskill in the art.

Example 23 RNA Interference Activity of Oligonucleotides

1. Dual-Luciferase Assay for RNA Interfering Evaluation

Modified RNAs obtained by solid-phase synthesis and click reactions wereevaluated in RNA interfering in dual-luciferase assays. Duplexes wereprepared by heating at 95° C. for 2 min and cooling to room temperaturefor 2 hours in 1X Phosphate buffered saline. (Table 12-14).

The ability of modified siRNAs to suppress gene expression was studiedby a dual-luciferase assay using a pGL4 vector (Promega), whichcontained the Renilla and firefly luciferase genes. The siRNA sequenceswere designed to target the firefly luciferase gene. Stably transfectedHeLa cells were then transfected with the indicated amounts of siRNAs,and the signals of firefly luciferase were normalized to those ofrenilla luciferase. HeLa SS6 cells were stably transfected with plasmidsencoding firefly (target) and Renilla (control) luciferases.

RNA interference methods: Hela cells were grown at 37° C., 5% CO₂ inDulbecco's modified Eagles's medium (DMEM, GIBCO) supplemented with 10%fetal bovine serum (FBS) and 1% antibiotic. The cells were maintained inthe exponential growth phase. The cells were then plated in 96-wellplates (0.1 mL medium per well) to reach about 90% confluence attransfection. Transfection was allowed to proceed for 24 hr at 37° C.and on day 2 the culture medium was changed to OPTIMEM 1, a reducedserum media (GIBCO). Two luciferase plasmids, Renilla luciferase(pRL-CMV) and firefly luciferase (pGL3) from Promega, were used ascontrol and reporter, respectively. Transfection of siRNAs was carriedout with Lipofectamine 2000 (Invitrogen) as described by themanufacturer for adherent cell lines. The final volume was 150 uL perwell. On day 2 cells are transfected with, firefly luciferase targeting,siRNA of varying concentrations ranging from 2 pM to 8 nM mediated byLipofectamine 2000 reagent (Invitrogen). After 24 h, cells were analyzedfor both firefly and renilla luciferase expression using a plateluminometer with a delay time of 2 s and an integrate time of 10 s.(VICTOR2, PerkinElmer, Boston, Mass.) and the Dual-Glo Luciferase Assaykit (Promega), FIG. 24. Firefly/renilla luciferase expression ratioswere used to determine percent gene silencing relative to untreated (nosiRNA) controls. Values generated represent the mean of triplicates.

The screening process began with a primary screen where all thecompounds were screened against the parent unmodified duplex as acomparison. Some of the potent duplexes from this screen which showed IC50 values several fold lower than that of the parent were then selectedfor a secondary screen or head to head comparison. All compounds wereseen to be more potent than the parent duplex. However, the ones thatshowed at least 5 fold improved potency were the duplexes with Uda (C22linker) at position 2, 15 and 16 on the sense strand (Table 15). Thefinal set of compounds was compared to one of our best modificationsAD3202 and it can be seen that these compounds match up to AD 3202 inpotency, however the exact mechanism by which the Uda modificationcontributes to improved activity is to be determined through furtherstudies.

Materials and Methods—

Cells and Reagents

Dual HeLa (HeLa Cells Transfected with PGL4 Plasmid for LuciferaseExpression)

Tissue culture medium, trypsin and Lipofectamine2000—purchased fromInvitrogen (Carlsbad, Calif.).

DMEM supplemented with 10% fetal bovine serum, 1% antibiotic solution.siRNA treatment was performed using Opti-MEM (Invitrogen) containing 1mg/ml Lipofectamine2000

DMEM without phenol red, Promega Dual Glo Luciferase Assay kit, 0.25%Trypsin (Invitrogen).

TABLE 12 Duplex information and IC50 values (Part I) Strand IC 50 ValuesDuplex No. S/AS Sequence 5′ to 3′ Modifications (nM) AD-1000 1000 SCUUACGCUGAGUACUUCGAdTdT None Artifact, 0.906 1001 ASUCGAAGUACUCAGCGUAAGdTdT None range 0.2-0.3 AD-20271 37079 SCUUACGCUGAG(T1y)ACUUCGAdTdT U12→(T1y) 0.22 1001 ASUCGAAGUACUCAGCGUAAGdTdT none AD-20272 37081 SCUUACGCUGAG(T31y)ACUUCGAdTdT U12→(T31y) 0.228 1001 ASUCGAAGUACUCAGCGUAAGdTdT none AD-20273 37083 S(T1y)CUUACGCUGAGUACUUCGAdTdT 5′ (T1y) 0.348 1001 ASUCGAAGUACUCAGCGUAAGdTdT none AD-20274 37085 S(T31y)CUUACGCUGAGUACUUCGAdTdT 5′ (T31y) 0.324 1001 ASUCGAAGUACUCAGCGUAAGdTdT none AD-20275 37087 S(u51y1)CUUACGCUGAGUACUUCGAdTdT 5′ (u51y1) 0.263 1001 ASUCGAAGUACUCAGCGUAAGdTdT none AD-20276 37089 S(u51y2)CUUACGCUGAGUACUUCGAdTdT 5′ (u51y2) 0.376 1001 ASUCGAAGUACUCAGCGUAAGdTdT none AD-20201 36862 SCUUACGCUGAGUACUUCGAdTdTL125 3′ L125 0.922 1001 ASUCGAAGUACUCAGCGUAAGdTdT none AD-20202 37075 SCUUACGCUGAGUACUUCGAdTdT(Tly) 3′ (T1y) 1.12 1001 ASUCGAAGUACUCAGCGUAAGdTdT none AD-20203 37077 SCUUACGCUGAGUACUUCGAdTdT(T31y) 3′ (T31y) 1.41 1001 ASUCGAAGUACUCAGCGUAAGdTdT none AD-20321 38103.1 SAAcGcuGGGcGuuAAucAAdTdTL125 2′OMe-all Py and 3′ out of range L125 24599AS UUGAUuAACGCCcAGCGUUdTsdT none

TABLE 13 Duplex information and IC50 values (Part II) Fold Increase inIC 50 activity as Strand Values compared to Duplex No. S/AS Sequence 5′to 3′ Modifications (nM) parent AD-1000 1000 S CUUACGCUGAGUACUUCGAdTdTNone 0.38 Parent 1001 AS UCGAAGUACUCAGCGUAAGdTdT None AD-20151 33024.1 SC(Uda)UACGCUGAGUACUUCGAdTdT U2→Uda 0.07 5 fold 1001 ASUCGAAGUACUCAGCGUAAGdTdT none AD-20152 33026.1 SCUUACGC(Uda)GAGUACUUCGAdTdT U8→Uda 0.176 2 fold 1001 ASUCGAAGUACUCAGCGUAAGdTdT none AD-20153 33027.1 SCUUACGCUGAG(Uda)ACUUCGAdTdT U12→Uda 0.044 8 fold 1001 ASUCGAAGUACUCAGCGUAAGdTdT none AD-20154 33028.1 SCUUACGCUGAGUAC(Uda)UCGAdTdT U15→Uda 0.092 4 fold 1001 ASUCGAAGUACUCAGCGUAAGdTdT none AD-20155 33029.1 SCUUACGCUGAGUACU(Uda)CGAdTdT U16→Uda 0.073 5 fold 1001 ASUCGAAGUACUCAGCGUAAGdTdT none AD-20156 33031.1 SCUUACGCUGAGUACUUCGAdT(Uda2p) Uda2p-3′end 1.18 1001 ASUCGAAGUACUCAGCGUAAGdTdT none AD-20157 33032.1 SCUUACGCUGAG(Uda)ACUUCGAdT(Uda2p) U12→Uda, NA-out of Uda2p -3′end range1001 AS UCGAAGUACUCAGCGUAAGdTdT none AD-20158 33033.1 SC(Uda)UACGCUGAGUACUUCGAdT(Uda2p) U2→Uda, 1.5 2 fold Uda2p-3′end 1001 ASUCGAAGUACUCAGCGUAAGdTdT none AD-20159 33034.1 SQ74CUUACGCUGAGUACUUCGAdTdT Q 74 - 5′end 0.154 2 fold 1001 ASUCGAAGUACUCAGCGUAAGdTdT none AD-20160 1000 S CUUACGCUGAGUACUUCGAdTdTnone 2.03 33035.1 AS UCGAAGUACUCAGCGUAAGdT(Uda2p) Uda2p-3′end-ASAD-20161 33071.1 S Q75CUUACGCUGAGUACUUCGAdTdT Q75-5′end 0.43 1001 ASUCGAAGUACUCAGCGUAAGdTdT none

TABLE 14 Duplex information and IC50 values (Part III) IC 50Fold Increase in Single Values activity as compared Duplex Strand # S/ASSequence 5′ to 3′ Modifications (nM) to parent AD-1000  1000 SCUUACGCUGAGUACUUCGAdTdT None 0.326 Parent  1001 ASUCGAAGUACUCAGCGUAAGdTdT None AD-20162 33558.1 SQ84CUUACGCUGAGUACUUCGAdTdT Q84 5′end 0.125 2.6 fold  1001 ASUCGAAGUACUCAGCGUAAGdTdT none AD-20163 33559.1 SQ85CUUACGCUGAGUACUUCGAdTdT Q85 5′end 0.126 2.6 fold  1001 ASUCGAAGUACUCAGCGUAAGdTdT none AD-20164 33560.1 SQ86CUUACGCUGAGUACUUCGAdTdT Q86 5′end 0.206 1.6 fold  1001 ASUCGAAGUACUCAGCGUAAGdTdT none AD-20165 33561.1 SQ87CUUACGCUGAGUACUUCGAdTdT Q87 5′end 0.35  1001 ASUCGAAGUACUCAGCGUAAGdTdT none AD-20166 33562.1 SQ88CUUACGCUGAGUACUUCGAdTdT Q88 5′end 0.318  1001 ASUCGAAGUACUCAGCGUAAGdTdT none AD-20167 33563.1a SQ89CUUACGCUGAGUACUUCGAdTdT Q89 5′end 0.771  1001 ASUCGAAGUACUCAGCGUAAGdTdT none AD-20168 33563.1b SQ89CUUACGCUGAGUACUUCGAdTdT Q89 5′end 0.953  1001 ASUCGAAGUACUCAGCGUAAGdTdT none

TABLE 15 Duplex information and IC50 values (Part IV) IC 50Fold Increase in Single Values activity as compared Duplex strand # S/ASSequence 5′ to 3′ Modifications (nM) to parent AD-1000 1000 SCUUACGCUGAGUACUUCGAdTdT None 0.242 1001 AS UCGAAGUACUCAGCGUAAGdTdT NoneAD-20151 33024.1 S C(Uda)UACGCUGAGUACUUCGAdTdT U2→Uda 0.036 6.5 fold1001 AS UCGAAGUACUCAGCGUAAGdTdT none AD-20152 33026.1 SCUUACGC(Uda)GAGUACUUCGAdTdT U8→Uda 0.083 3 fold 1001 ASUCGAAGUACUCAGCGUAAGdTdT none AD-20153 33027.1 SCUUACGCUGAG(Uda)ACUUCGAdTdT U12→Uda 0.052 4 fold 1001 ASUCGAAGUACUCAGCGUAAGdTdT none AD-20154 33028.1 SCUUACGCUGAGUAC(Uda)UCGAdTdT U15→Uda 0.044 5 fold 1001 ASUCGAAGUACUCAGCGUAAGdTdT none AD-20155 33029.1 SCUUACGCUGAGUACU(Uda)CGAdTdT U16→Uda 0.046 6 fold 1001 ASUCGAAGUACUCAGCGUAAGdTdT none AD-20162 33558.1 SQ84CUUACGCUGAGUACUUCGAdTdT Q84 5′end 0.074 3 fold 1001 ASUCGAAGUACUCAGCGUAAGdTdT none AD-20163 33559.1 SQ85CUUACGCUGAGUACUUCGAdTdT Q85 5′end 0.062 4 fold 1001 ASUCGAAGUACUCAGCGUAAGdTdT none

1. An oligonucleotide comprising at least one subunit of formula (I) atone or more positions that occur at 1-6 nucleotides from either end ofthe oligonucleotide:

wherein: X is O, S, NR^(N) or CR^(P) ₂; B is hydrogen, optionallysubstituted natural or non-natural nucleobase, optionally substitutedtriazole, or optionally substituted tetrazole, NH—C(O)—O—C(CH₂B₁)₃,NH—C(O)—NH—C(CH₂B₁)₃; where B₁ is independently halogen, mesylate, N₃,CN, optionally substituted triazole or optionally substituted tetrazole,and where the nucleobase may further be substituted by -J-linker-N₃,-J-linker-CN, -J-linker-cycloalkyne, -J-linker-R^(L), -Linker-Q-R^(L),or -J-linker-Q-linker-R^(L); R¹, R², R³, R⁴ and R⁵ are eachindependently H, OR⁶, F, N(R^(N))₂, N₃, CN, -J-linker-N₃, -J-linker-CN,-J-linker-C≡R⁸, -J-linker-cycloalkyne, -J-linker-R^(L), -Linker-Q-R^(L),or -J-linker-Q-linker-R^(L); J is absent, O, S, NR^(N), OC(O)NH,NHC(O)O, C(O)NH, NHC(O), NHSO, NHSO₂, NHSO₂NH, OC(O), C(O)O, OC(O)O,NHC(O)NH, NHC(S)NH, OC(S)NH, OP(N(R^(P))₂)O, or OP(N(R^(P))₂); R⁶ ishydrogen, hydroxyl protecting group, optionally substituted alkyl,optionally substituted aryl, optionally substituted cycloalkyl,optionally substituted aralkyl, optionally substituted alkenyl,optionally substituted heteroaryl, polyethyleneglycol (PEG), aphosphate, a diphosphate, a triphosphate, a phosphonate, aphosphonothioate, a phosphonodithioate, a phosphorothioate, aphosphorothiolate, a phosphorodithioate, a phosphorothiolothionate, aphosphodiester, a phosphotriester, an activated phosphate group, anactivated phosphite group, a phosphoramidite, a solid support,—P(Z¹)(Z²)—O-nucleoside, —P(Z¹)(Z²)—O-oligonucleotide,—P(Z¹)(Z²)-formula (I), —P(Z¹)(O-linker-Q-linker-R^(L))—O-nucleoside,P(Z¹)(O-linker-R^(L))—O-nucleoside, —P(Z¹)(O-linker-N₃)—O-nucleoside,P(Z′)(O-linker-CN)—O-nucleoside, P(Z¹)(O-linker-C≡R⁸)—O-nucleoside,P(Z¹)(O-linker-cycloalkyne)-O-nucleoside,—P(Z¹)(O-linker-Q-linker-R^(L))—O-oligonucleotide,P(Z¹)(O-linker-R^(L))—O-oligonucleotide,P(Z¹)(O-linker-N₃)—O-oligonucleotide,—P(Z¹)(O-linker-CN)—O-oligonucleotide,P(Z¹)(O-linker-C≡R⁸)—O-oligonucleotide,P(Z¹)(O-linker-cycloalkyne)-O-oligonucleotide,—P(Z¹)(-linker-Q-linker-R^(L))—O-nucleoside,—P(Z¹)(-linker-Q-R^(L))—O-nucleoside, —P(Z¹)(-linker-N₃)—O-nucleoside,P(Z¹)(-linker-CN)—O-nucleoside, P(Z¹)(-linker-C≡R⁸)—O-nucleoside,P(Z¹)(-linker-cycloalkyne)-O-nucleoside,—P(Z¹)(-linker-Q-linker-R^(L))—O-oligonucleotide,—P(Z¹)(-linker-R^(L))—O-oligonucleotide,P(Z¹)(-linker-N₃)—O-oligonucleotide,—P(Z¹)(-linker-CN)—O-oligonucleotide,P(Z¹)(-linker-C≡R⁸)—O-oligonucleotide orP(Z¹)(-linker-cycloalkyne)-O-oligonucleotide; R^(N) is H, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted aryl, optionally substitutedcycloalkyl, optionally substituted aralkyl, optionally substitutedheteroaryl, or an amino protecting group; R^(P) is independently H,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted aryl, optionally substitutedcycloalkyl, or optionally substituted heteroaryl; Q is

R^(L) is hydrogen or a ligand; R⁸ is N or CR⁹; R⁹ is H, optionallysubstituted alkyl, or silyl; Z¹ and Z² are each independently O, S, oroptionally substituted alkyl; provided that Q, —N₃, —CN, —C≡R⁸, anoptionally substituted triazole, or an optionally substituted tetrazoleis present at least once in the compound.
 2. (canceled)
 3. Theoligonucleotide of claim 1, comprising at least one subunit representedby formula (III):

wherein R₁, R₂, R₃, R₄, R₅ and B are as previously defined in claim 1.4. The oligonucleotide of claim 1, comprising at least one subunitrepresented by formula (IV):

wherein R¹⁰ and R²⁰ are each independently hydrogen, optionallysubstituted aliphatic, optionally substituted aryl, or optionallysubstituted heteroaryl; and wherein R¹, R², R³, R⁴, R⁵, and X are asdefined in claim
 1. 5. The oligonucleotide of claim 1, comprising atleast one subunit represented by formula (V):

wherein B₁ is halogen, N₃, CN, optionally substituted triazole, oroptionally substituted tetrazole; R¹, R², R³, R⁴, and R⁵ are as definedin claim
 1. 6. The oligonucleotide of claim 1, wherein the ligand isselected from the group consisting of thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine, multivalentN-acetyl-galactosamine, N-acetyl-glucosamine, multivalent N-acetylglucosamine, multivalent mannose, multivalent fucose, glycosylatedpolyaminoacids, mannose, lactose, galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, and an RGD peptide. 7.(canceled)
 8. The oligonucleotide of claim 1, wherein the linker isrepresented by structure—[P-Q₁-R]_(q)-T-, wherein: P, R and T are each independently absent, CO,NH, O, S, OC(O), NHC(O), CH₂, CH₂NH, CH₂O; NHCH(R^(a))C(O),—C(O)—CH(R^(a))—NH—, —C(O)-(optionally substituted alkyl)-NH—, CH═N—O,

Q₁ is absent, —(CH₂)_(n)—, —C(R¹⁰⁰)(R²⁰⁰)(CH₂)_(n)—,—(CH₂)_(n)C(R¹⁰⁰)(R²⁰⁰)—, —(CH₂CH₂O)_(m)CH₂CH₂—, or—(CH₂CH₂O)_(m)CH₂CH₂NH—; R^(a) is H or an amino acid side chain; R¹⁰⁰and R²⁰⁰ are each independently H, CH₃, OH, SH or N(R^(X))₂; R^(X) isindependently H, methyl, ethyl, propyl, isopropyl, butyl or benzyl; q isan integer from 0-20; n is an integer from 1-20; and m is an integerfrom 0-50; provided that at least one of P, R, T, and Q₁ is present inthe linker. 9-11. (canceled)
 12. The oligonucleotide of claim 1, whereinthe oligonucleotide is a single-stranded oligonucleotide.
 13. Theoligonucleotide of claim 12, wherein the single-stranded oligonucleotideis a single-stranded siRNA.
 14. The oligonucleotide of claim 12, whereinthe single-stranded oligonucleotide is a microRNA.
 15. Theoligonucleotide of claim 1, wherein the oligonucleotide is adouble-stranded oligonucleotide.
 16. The oligonucleotide of claim 15,wherein the double-stranded oligonucleotide is a double-stranded siRNA.17-20. (canceled)
 21. A pharmaceutical composition comprising theoligonucleotide of claim 1 and a pharmaceutically acceptable excipient.22-24. (canceled)
 25. The oligonucleotide of claim 1, wherein R¹ and R⁴are H.
 26. The oligonucleotide of claim 1, wherein R⁵ is OR⁶.
 27. Theoligonucleotide of claim 1, wherein R³ is OR⁶; and R² is—O-Linker-Q-Linker-R^(L), —OC(O)N(H)-Linker-Q-Linker-R^(L), or-Linker-Q-Linker-R^(L).
 28. The oligonucleotide of claim 27, wherein R²is

and f is 1-20.
 29. The oligonucleotide of claim 28, wherein R² is


30. The oligonucleotide of claim 6, wherein the ligand is mannose,lactose, galactose, N-acetyl-galactosamine, N-acetyl glucosamine,multivalent lactose, multivalent galactose, multivalent mannose,multivalent fucose, multivalent N-acetyl-galactosamine, or multivalentN-acetyl glucosamine.
 31. The oligonucleotide of claim 1, wherein atleast two subunits of formula I is incorporated at positions that occurat 1-6 nucleotides from either end of the oligonucleotide.
 32. Theoligonucleotide of claim 1, wherein at least three subunits of formula Iis incorporated at positions that occur at 1-6 nucleotides from eitherend of the oligonucleotide.